Luminophore-labeled molecules coupled with particles for microarray-based assays

ABSTRACT

A method for labeling target molecules coupled to particles for the detection of the target molecules using a microarray chip, comprises: providing a functionalized microparticle, wherein the microparticle is coated with one or more functional group; providing a modification group on each of the target molecules to be detected to form modified target molecules; contacting the functionalized microparticle with the modified target molecules; coupling a luminophore to the complex between the functionalized microparticle and the modified target molecules, thereby directly or indirectly labeling each modified target molecules with the luminophore. By directly or indirectly labeling the target molecules with the luminophore, the method reduces the cost of fluorescence detection, and avoids PCR inhibition derived from traditional fluorescence labeling molecules.

CROSS REFERENCE TO RELATED APPLICATIONS

The present application is a U.S national phase of International PatentApplication No. PCT/CN2014/001085, filed Dec. 2, 2014, which claimspriority benefit to Chinese Patent Application No. 201310651906.5, filedon Dec. 5, 2013, published as Chinese Patent No. CN 103760355B on Sep.16, 2014, the disclosures of which applications are incorporated byreference herein in their entireties for all purposes.

TECHNICAL FIELD

In certain aspects, the present disclosure relates to methods forlabeling target molecules for detection using a microarray chip. Forexample, the target molecules can be nucleic acid molecules coupled to aparticle. In particular, the present disclosure relates tomicroarray-based methods, compositions, and kits for analyzing molecularinteractions, e.g., multiplexed genetic analysis of nucleic acidfragments, for diagnosis of clinical samples and disease-associatedtesting.

SUBMISSION OF SEQUENCE LISTING ON ASCII TEXT FILE

The content of the following submission on ASCII text file isincorporated herein by reference in its entirety: a computer readablefrom (CRF) of the Sequence Listing (file name: 514572008200SEQLIST.txt,date recorded: May 27, 2016, size: 6,150 bytes).

BACKGROUND

In recently years, microarray technologies enable the evaluation of upto tens of thousands of molecular interactions simultaneously in ahigh-throughput manner. DNA microarray-based assays have been widelyused, including the applications for gene expression analysis,genotyping for mutations, single nucleotide polymorphisms (SNPs), andshort tandem repeats (STRs), with regard to drug discovery, diseasediagnostics, and forensic purpose (Heller, Ann Rev Biomed Eng (2002) 4:129-153; Stoughton, Ann Rev Biochem (2005) 74: 53-82; Hoheisel, Nat RevGenet (2006) 7: 200-210). Pre-determined specific oligonucleotide probesimmobilized on microarray can serve as a de-multiplexing tool to sortspatially the products from parallel reactions performed in solution(Zhu et al., Antimicrob Agents Chemother (2007) 51: 3707-3713), and evencan be more general ones, e.g., the designed and optimized artificialtags or their complementary sequences employed in the universalmicroarray (Gerrey et al., J Mol Biol (1999) 292: 251-262; Li et al.,Hum Mutat (2008) 29: 306-314). Combined with the multiplex PCR method,microarray-based assays for SNPs and gene mutations, such as deletions,insertions, and indels, thus can be carried out in routine genetic anddiagnostic laboratories.

Meanwhile, protein and chemical microarrays have emerged as twoimportant tools in the field of proteomics (Xu and Lam, J BiomedBiotechnol (2003) 5: 257-266). Specific proteins, antibodies, smallmolecule compounds, peptides, and carbohydrates can now be immobilizedon solid surfaces to form microarrays, just like DNA microarrays. Thesearrays of molecules can then be probed with simple composition ofmolecules or complex analytes.

Interactions between the analytes and the immobilized array of moleculesare evaluated with a number of different detection systems. Typically,commercial use of microarrays employs optical detection withfluorescent, chemiluminescent or enzyme labels, electrochemicaldetection with enzymes, ferrocene or other electroactive labels, as wellas label-free detection based on surface plasmon resonance ormicrogravimetric techniques (Sassolas et al., Chem Rev (2008) 108:109-139). To further simplify the assay protocol and reduce the relianceon related equipment, magnetic bead labeling was employed so that assayresults could be photographed with a charge-coupled device (CCD)assisted camera or viewed under low magnification microscope (Guo etal., J Anal Sci (2007) 23: 1-4; Li et al., supra; Shlyapnikov et al.,Anal Biochem (2010) 399: 125-131), and cross-reactive contacts orunspecific bonds even can be quickly eliminated by applying magneticfield or shear flow (Mulvaney et al., Anal Biochem (2009) 392: 139-144).The detection of microarray-hybridized DNA with magnetic beads thusopens a new way to routine hybridization assays which do not requireprecise measurements of DNA concentration in solution.

Typically, a fluorescent marker is used to label each target moleculefor it to be detected within a plurality of molecules. In these cases,the primers in the PCR reaction often need to be modified by chemical orfluorescent modifications for each target molecule, which can lead tohigh cost and inhibition of the PCR reaction. There is therefore a needfor methods, compositions, and kits for labeling target molecules in amicroarray assay that address the above issues and related needs.

SUMMARY

In one aspect, disclosed herein is a method of labeling a plurality oftarget molecules with a luminophore for detecting the target moleculesusing a microarray. In some embodiments, the method comprises: couplingeach target molecule of a plurality of target molecules to a particle toform a target-particle complex, wherein each target molecule comprises amodification moiety and the particle comprises a plurality of functionalmoieties, wherein each target molecule is coupled to the particle viainteraction between the modification moiety and the functional moiety,and wherein each target molecule comprises a target portion and aportion capable of specific binding to a probe molecule immobilized on amicroarray, wherein the microarray comprises a plurality of immobilizedprobe molecules; and providing a luminophore on the target-particlecomplex, thereby directly or indirectly labeling the plurality of targetmolecules with the luminophore. In particular embodiments, the providingstep comprises: labeling a subset of the plurality of target moleculeswith the luminophore, and incubating the plurality of target moleculeswith the particle, whereby the plurality of target molecules are coupledto the particle, wherein the target-particle complex comprises targetmolecule(s) labeled with the luminophore and target molecule(s) notlabeled with the luminophore; and/or labeling a subset of the functionalmoieties of the particle with the luminophore, and incubating theluminophore-labeled particle with the plurality of target molecules,whereby the plurality of target molecules are coupled to the particle;and/or introducing the luminophore into or onto the particle to labelthe particle, and incubating the luminophore-labeled particle with theplurality of target molecules, whereby the plurality of target moleculesare coupled to the particle; and/or providing a labeling moleculecomprising: (1) the luminophore, and (2) the modification moiety capableof interacting with the functional moiety of the particle, wherein thelabeling molecule does not bind to the immobilized probe molecule on themicroarray, and incubating the labeling molecule with the plurality oftarget molecules and the particle, whereby the labeling molecule and theplurality of target molecules are coupled to the particle; and/orproviding a binding molecule comprising the luminophore, wherein thebinding molecule is capable of specific binding to a subset of theplurality of target molecules, and wherein the binding molecule does notbind to the immobilized probe molecule on the microarray or directly tothe particle, and incubating the binding molecule with the plurality oftarget molecules and the particle, whereby the plurality of targetmolecules are coupled to the particle.

In another aspect, the present disclosure provides a method of labelinga target molecule with a luminophore for detecting the target moleculeusing a microarray, the method comprising: coupling a target molecule toa particle to form a target-particle complex, wherein the targetmolecule comprises a modification moiety and the particle comprises afunctional moiety, wherein the target molecule is coupled to theparticle via interaction between the modification moiety and thefunctional moiety, and wherein the target molecule comprises a targetportion and a portion capable of specific binding to a probe moleculeimmobilized on a microarray; and providing a luminophore on thetarget-particle complex, thereby directly or indirectly labeling thetarget molecule with the luminophore. In one aspect, the target moleculecomprises a plurality of target molecules, and the microarray comprisesa plurality of immobilized probe molecules. In another aspect, theparticle comprises a plurality of functional moieties, each of which iscapable of interacting with the modification moiety of the targetmolecule, and a plurality of target molecules are coupled to theparticle.

In any of the preceding embodiments, each of the plurality of targetmolecules can comprise a modification moiety capable of interacting withthe functional moiety of the particle. In some aspects, the portion ofthe target molecule capable of specific binding to the immobilized probemolecule comprises a nucleotide sequence.

In any of the preceding embodiments, the method can further comprise astep of detecting the target molecule using the microarray. In oneaspect, the detecting step comprises measuring luminescence of thetarget-particle complex, wherein the complex is immobilized on themicroarray via specific binding of the target molecule to theimmobilized probe molecule.

In some embodiments, the providing step comprises labeling a subset ofthe plurality of target molecules with the luminophore, and incubatingthe plurality of target molecules with the particle, whereby theplurality of target molecules are coupled to the particle, wherein thetarget-particle complex comprises target molecule(s) labeled with theluminophore and target molecule(s) not labeled with the luminophore,whereby the plurality of target molecules are directly or indirectlylabeled with the luminophore.

In one embodiment, the providing step comprises labeling a subset of thefunctional moieties of the particle with the luminophore, and incubatingthe luminophore-labeled particle with the plurality of target molecules,whereby the plurality of target molecules are coupled to the particle,whereby the plurality of target molecules are indirectly labeled withthe luminophore.

In some embodiments, the providing step comprises introducing theluminophore into or onto the particle to label the particle, andincubating the luminophore-labeled particle with the plurality of targetmolecules, whereby the plurality of target molecules are coupled to theparticle, whereby the plurality of target molecules are indirectlylabeled with the luminophore.

In other embodiments, the providing step comprises providing a labelingmolecule comprising: (1) the luminophore, and (2) the modificationmoiety capable of interacting with the functional moiety of theparticle, wherein the labeling molecule does not bind to the immobilizedprobe molecule on the microarray, and incubating the labeling moleculewith the plurality of target molecules and the particle, whereby thelabeling molecule and the plurality of target molecules are coupled tothe particle, whereby the plurality of target molecules are indirectlylabeled with the luminophore. In one aspect, the labeling molecule doesnot contain the target portion of the target molecule. In anotheraspect, the labeling molecule does not bind to the plurality of targetmolecules.

In still other embodiments, the providing step comprises providing abinding molecule comprising the luminophore, wherein the bindingmolecule is capable of specific binding to a subset of the plurality oftarget molecules, and wherein the binding molecule does not bind to theimmobilized probe molecule on the microarray or directly to theparticle, and incubating the binding molecule with the plurality oftarget molecules and the particle, whereby the plurality of targetmolecules are coupled to the particle, whereby the plurality of targetmolecules are indirectly labeled with the luminophore.

In any of the preceding embodiments, the target molecule can comprise apolynucleotide, a polypeptide, an antibody, a small molecule compound, apeptide, a carbohydrate, or a combination thereof. In one aspect, thetarget molecule is a polynucleotide enriched by an amplificationreaction such as PCR.

In any of the preceding embodiments, the functional moiety can beselected from the group consisting of a chemical group, apolynucleotide, a polypeptide, an antibody, a small molecule compound, apeptide, and a carbohydrate. In one aspect, the chemical group is analdehyde, hydroxyl, carboxyl, ester, amine, sulfo, or sulthydryl group.

In any of the preceding embodiments, the functional moiety can bestreptavidin, neutravidin, or avidin. In any of the precedingembodiments, the luminophore can be a fluorophore, a phosphorescentmoiety, or a chromophore. In any of the preceding embodiments, themodification moiety can be biotin, digoxin, digoxigenin, apolynucleotide, a poly-dA, apoly-dT, a protein, a polypeptide, or acarbohydrate. In any of the preceding embodiments, the luminophore canbe a quantum dot, a luminescent protein, a green fluorescent protein(GFP), or a small molecule fluorescent dye. In some embodiments, thesmall molecule fluorescent dye is selected from the group consisting of:a xanthene derivative, fluorescein, rhodamine, Oregon green, eosin,texas red; a cyanine derivative, cyanine, indocarbocyanine,oxacarbocyanine, thiacarbocyanine, merocyanine; a naphthalenederivative, a dansyl derivative, a prodan derivative, a dansyl andprodan derivative; a coumarin derivative; a thiadiazole derivative; anoxadiazole derivative, pyridyloxazole, nitrobenzoxadiazole,benzoxadiazole; a pyrene derivative, cascade blue; BODIPY (Invitrogen);an oxazine derivative, Nile red, Nile blue, cresyl violet, oxazine 170;an acridine derivative, proflavin, acridine orange, acridine yellow; anarylmethine derivative, auramine, crystal violet, malachite green; a CFdye (Biotium), an Alexa Fluor dye (Invitrogen), Atto and Tracy (Sigma),a Tetrapyrrole derivative, porphin, phtalocyanine, bilirubin, cascadeyellow, azure B, acridine orange, DAPI, Hoechst 33258, lucifer yellow,piroxicam, quinine, anthraqinone, squarylium, and oligophenylenes.

In any of the preceding embodiments, the luminophore can be a compoundof a transition metal or a rare earth compound. In any of the precedingembodiments, the luminophore can be a food coloring agent, a fabric dye,lycopene, β-carotene, an anthocyanin, chlorophyll, hemoglobin,hemocyanin, or a mineral. In one aspect, the fabric dye is an azocompound. In one aspect, the mineral is malachite or amethyst.

In any of the preceding embodiments, the particle can be amicroparticle. In any of the preceding embodiments, the particlediameter can be between about 0.1 micrometers and about 10 micrometers,about 0.1 micrometers and about 0.5 micrometers, about 0.5 micrometersand about 1 micrometer, about 1 micrometer and about 2 micrometers,about 2 micrometers and about 4 micrometers, about 4 micrometers andabout 6 micrometers, about 6 micrometers and about 8 micrometers, orabout 8 micrometers and about 10 micrometers. In some embodiments, theparticle diameter is less than about 0.1 micrometers, or more than about10 micrometers. In some embodiments, the particle is a magnetic particleor a paramagnetic particle. In some aspects, the particle is aparamagnetic microsphere.

In any of the preceding embodiments, the immobilized probe molecule onthe microarray can comprise a polynucleotide, a protein, a polypeptide,or a carbohydrate. In some aspects, the microarray comprises a substratecomprising silicon, glass, plastic, hydrogel, agarose, nitrocellulose,nylon, or a combination thereof.

In another aspect, provided herein is a method of detecting a targetmolecule using a microarray, the method comprising: labeling a targetmolecule with a luminophore according to any of the precedingembodiments; incubating the target molecule and the particle with themicroarray, wherein the target-particle complex is immobilized on themicroarray via specific binding of the target molecule to theimmobilized probe molecule; and measuring luminescence of theimmobilized target-particle complex, wherein the luminescence indicatesthe absence, presence, and/or amount of the target molecule. In oneaspect, the method further comprises identifying the target portion ofthe target molecule based on the immobilized probe molecule on themicroarray.

In any of the preceding embodiments, the target molecule can beassociated with a disease caused by an infectious or pathogenic agentselected from the group consisting of a fungus, a bacterium, amycoplasma, a rickettsia, a chlamydia, a virus, and a protozoa. In anyof the preceding embodiments, the target molecule can be associated witha sexually transmitted disease, cancer, cerebrovascular disease, heartdisease, respiratory disease, coronary heart disease, diabetes,hypertension, Alzheimer's disease, neurodegenerative disease, chronicobstructive pulmonary disease, autoimmune disease, cystic fibrosis,spinal muscular atrophy, thalassemia (such as alpha-thalassemia,beta-thalassemia, and delta-thalassemia), phenylalanine hydroxylasedeficiency, Duchenne muscular dystrophy, or hereditary hearing loss.

In any of the preceding embodiments, the probe molecule can comprise apolynucleotide, a polypeptide, an antibody, a small molecule compound, apeptide, a carbohydrate, or a combination thereof. In any of thepreceding embodiments, the microarray can comprise at least two probemolecules, for example, at least about 5, about 10, about 50, about 100,about 1,000, about 10⁴, about 10⁵, about 10⁵, about 10⁶, about 10⁷,about 10⁸, about 10⁹, about 10¹⁰, or more than about 10¹⁰ probemolecules.

In any of the preceding embodiments, the microarray can be fabricatedusing a technology selected from the group consisting of printing with afine-pointed pin, photolithography using a pre-made mask,photolithography using a dynamic micromirror device, ink-jet printing,microcontact printing, and electrochemistry on a microelectrode array.In any of the preceding embodiments, a spot on the microarray can rangefrom about 1 micrometer to about 5000 micrometers in diameter. In someaspects, the spot can range from about 1 micrometer to about 10micrometers, about 10 micrometers to about 100 micrometers, about 100micrometers to about 500 micrometers, about 500 micrometers to about1000 micrometers, about 1000 micrometers to about 2500 micrometers, orabout 2500 micrometers to about 5000 micrometers in diameter.

In any of the preceding embodiments, the probe molecule can be attachedto the microarray by in situ synthesis, nonspecific adsorption, specificbinding, nonspecific chemical ligation, chemoselective ligation, orcovalent binding. In any of the preceding embodiments, the interactionbetween the target molecule and the probe molecule can be anon-covalent, reversible covalent or irreversible covalent interaction.

In any of the preceding embodiments, the efficiency and/or efficacy ofthe interaction between the target molecule and the probe molecule canbe enhanced by an external force. In some aspects, the external force isa magnetic force, a dielectrophoretic force, a mechanical force, or acombination thereof.

In any of the preceding embodiments, the target molecule can be subjectto an in vitro manipulation, for example, laser treatment,ultrasonication treatment, heat treatment, microwave treatment,piezoelectricity treatment, electrophoresis, dielectrophoresis, solidphase adhesion, filtration, fluidic stress, enzymatic digestion, PCRamplification, reverse-transcription, reverse-transcription PCRamplification, allele-specific PCR (ASPCR), single-base extension (SBE),allele specific primer extension (ASPE), restriction enzyme digestion,strand displacement amplification (SDA), transcription mediatedamplification (TMA), ligase chain reaction (LCR), nucleic acid sequencebased amplification (NASBA), primer extension, rolling circleamplification (RCA), self sustained sequence replication (3SR), the useof Q Beta replicase, nick translation, or loop-mediated isothermalamplification (LAMP), or any combination of the in vitro manipulationsdisclosed herein.

In any of the preceding embodiments, the target molecule can comprise adouble-stranded polynucleotide and/or a single stranded polynucleotide.In one aspect, the target molecules is a double-stranded polynucleotideand is denatured to become single-stranded by a chemical reaction, anenzyme, heating, or a combination thereof, before or after coupling tothe particle. In one aspect, the enzyme is an exonuclease, aUracil-N-glycosylase, or a combination thereof. In one aspect, thechemical reaction uses urea, formamide, methanol, ethanol, sodiumhydroxide, or a combination thereof. In another aspect, thedouble-stranded target polynucleotide is denatured at an appropriatetemperature from about 30° C. to about 95° C., about 30° C. to about 35°C., about 35° C. to about 40° C., about 40° C. to about 45° C., about45° C. to about 50° C., about 50° C. to about 55° C., about 55° C. toabout 60° C., about 60° C. to about 65° C., about 65° C. to about 70°C., about 70° C. to about 75° C., about 75° C. to about 80° C., about80° C. to about 85° C., about 85° C. to about 90° C., or about 90° C. toabout 95° C.

In any of the preceding embodiments, the target molecule can be apolynucleotide and can be coupled to the particle through astreptavidin/biotin interaction, a neutravidin/biotin interaction, anavidin/biotin interaction, or a poly-dT/dA interaction.

In any of the preceding embodiments, the target molecule can comprise auniversal tag sequence. In one aspect, the target molecule is apolynucleotide of a species, and wherein the universal tag sequence haslow homology to the genomic DNA of the species. In another aspect, thetag sequence has no hair-pin structure. In any of the precedingembodiments, the tag sequence can be a single stranded oligonucleotideor modified analog.

In any of the preceding embodiments, the tag sequence can be a lockednucleic acid (LNA), a Zip nucleic acid (ZNA), or a peptide nucleic acid(PNA). In any of the preceding embodiments, at least two differentuniversal tag sequences can be used. In one aspect, the T_(m) differencebetween different tag sequences equals or is less than about 5° C. Insome aspects, the T_(m) difference is about 0.1° C., about 0.5° C.,about 1° C., about 1.5° C., about 2° C., about 2.5° C., about 3° C.,about 3.5° C., about 4° C., about 4.5° C., or about 5° C. In anotheraspect, different tag sequences have no cross-hybridization amongthemselves.

In any of the preceding embodiments, the tag sequence can be introducedto the target molecule during an in vitro manipulation.

In any of the preceding embodiments, the method can further comprisedetecting the target molecule by a microarray scanning device, anordinary image-capturing device, or a naked eye. In some aspects, themicroarray scanning device employs optical detection with a fluorescentlabel, a chemiluminescent label, a phosphorescent label, or achromophore label. In one embodiment, the microarray scanning deviceemploys one or more detection methods based on surface plasmonresonance, magnetic force, giant magnetoresistance, or microgravimetrictechnique. In one embodiment, the ordinary image-capturing device is aflatbed scanner, a camera, or a portable device. In one aspect, thecamera is with or without the assistance of a lens, a magnifier, or amicroscope. In one aspect, the portable device is a camera on a mobilephone or a laptop computer with or without the assistance of a lens, amagnifier, or a microscope.

In any of the preceding embodiments, the target molecule can be a singlestranded polynucleotide. In one aspect, the complementary strand of thetarget polynucleotide is labeled.

In any of the preceding embodiments, the target molecule can beassociated with a genetic information, for example, a substitution, aninsertion, a deletion, an indel, or any combination thereof. In oneaspect, the genetic information is a single nucleotide polymorphism(SNP).

In any of the preceding embodiments, the genetic information can beassociated with a disease caused by an infectious or pathogenic agentselected from the group consisting of a fungus, a bacterium, amycoplasma, a rickettsia, a chlamydia, a virus, and a protozoa. In anyof the preceding embodiments, the genetic information can be associatedwith a sexually transmitted disease, cancer, cerebrovascular disease,heart disease, respiratory disease, coronary heart disease, diabetes,hypertension, Alzheimer's disease, neurodegenerative disease, chronicobstructive pulmonary disease, autoimmune disease, cystic fibrosis,spinal muscular atrophy, beta thalassemia, phenylalanine hydroxylasedeficiency, Duchenne muscular dystrophy, or hereditary hearing loss. Inone aspect, the genetic information is associated with hereditaryhearing loss. In some embodiments, the genetic information is within atarget gene of GJB2 (Cx26), GJB3 (Cx31), SLC26A4 (PDS), 12S rRNA(MTRNR1), or a β-globin gene such as HBB. In some aspects, the geneticinformation in GJB2 is selected from the group consisting of c.35delG,c.176_191del16, c.235delC, and c.299_300delAT. In one aspect, thegenetic information in SLC26A4 is selected from the group consisting ofc.2168A>G, IVS7-2A>G (c.919-2A>G), c.1229C>T, c.1975G>C, c.1174A>T,c.1226G>A, c.2027T>A, and IVS15+5G>A. In one aspect, the geneticinformation in GJB3 (Cx31) is c.538 C>T. In another aspect, the geneticinformation in 12S rRNA is selected from the group consisting ofm.1494C>T and m.1555A>G. In one aspect, the genetic information isassociated with beta thalassemia. In some embodiments, the geneticinformation is within a target gene of HBB. In some aspects, the geneticinformation in HBB is selected from the group consisting of c.-82C>A,c.-80T>C, c.-79A>G, c.-78A>G, c.-11_8delAAAC, c.79G>A, c.91A>G,c.92+1G>T, c.92+5G>C, c.315+5G>C, c.316-197C>T, c.2T>G, c.45_46insG,c.84_85insC, c.52A>T, c.113G>A, c.126_129delCTTT, c.130G>T, andc.216_217insA.

In any of the preceding embodiments, ASPCR can be used to amplify thegenetic information. In one aspect, the set of primers for the ASPCRcomprises at least two allele-specific primers and one common primer. Inone embodiment, the allele-specific primers and the common primer have asequence as set forth in Table 2. In another embodiment, theallele-specific primers terminate at the SNP/mutation locus.

In any of the preceding embodiments, the allele-specific primers canfurther comprise an artificial mismatch to the corresponding targetsequence. In any of the preceding embodiments, the allele-specificprimers can comprise a natural nucleotide or analog thereof. In any ofthe preceding embodiments, the allele-specific primers can comprise atag sequence.

In any of the preceding embodiments, the ASPCR can use a DNA polymerasewithout the 3′ to 5′ exonuclease activity. In any of the precedingembodiments, genetic information of at least two genetic loci can bedetected. In one aspect, multiplex PCR is used to amplify the geneticinformation of the at least two genetic loci.

In any of the preceding embodiments, the genetic material for ASPCR canbe isolated from tissues, cells, body fluids, hair, nail and ejaculate,including saliva sample, sputum sample, sperm sample, oocyte sample,zygote sample, lymph sample, blood sample, interstitial fluid sample,urine sample, buccal swab sample, chewing gum sample, cigarette buttsample, envelope sample, stamp sample, prenatal sample, or dried bloodspot sample.

In one aspect, disclosed herein is a composition comprising: a pluralityof target molecules, a subset of which is labeled with a luminophore,wherein each of the target molecules comprises a modification moiety anda portion capable of specific binding to an immobilized probe moleculeon a microarray; and a particle comprising a plurality of functionalmoieties capable of interacting with the modification moieties of thetarget molecules, wherein the plurality of target molecules are coupledto the particle via interaction between the modification moieties andthe functional moieties, and wherein the plurality of target moleculesare directly or indirectly labeled with the luminophore.

In another aspect, disclosed herein is a composition comprising: aplurality of target molecules, wherein each of the target moleculescomprises a modification moiety and a portion capable of specificbinding to an immobilized probe molecule on a microarray; and a particlecomprising a plurality of functional moieties capable of interactingwith the modification moieties of the target molecules, a subset ofwhich functional moieties of the particle is labeled with a luminophore,wherein the plurality of target molecules are coupled to the particlevia interaction between the modification moieties and the functionalmoieties, and wherein the plurality of target molecules are indirectlylabeled with the luminophore.

In still another aspect, disclosed herein is a composition comprising: aplurality of target molecules, wherein each of the target moleculescomprises a modification moiety and a portion capable of specificbinding to an immobilized probe molecule on a microarray; and a particlecomprising a plurality of functional moieties capable of interactingwith the modification moieties of the target molecules, wherein theparticle comprises a luminophore introduced therein, wherein theplurality of target molecules are coupled to the particle viainteraction between the modification moieties and the functionalmoieties, and wherein the plurality of target molecules are indirectlylabeled with the luminophore.

In still another aspect, disclosed herein is a composition comprising: aplurality of target molecules, wherein each of the target moleculescomprises a modification moiety and a portion capable of specificbinding to an immobilized probe molecule on a microarray; a particlecomprising a plurality of functional moieties capable of interactingwith the modification moieties of the target molecules; and a labelingmolecule comprising a luminophore, wherein the labeling molecule iscapable of interacting with the functional moiety of the particle,wherein the labeling molecule does not bind to the immobilized probemolecule on the microarray, wherein the labeling molecule and theplurality of target molecules are coupled to the particle viainteraction between the modification moieties and the functionalmoieties, and wherein the plurality of target molecules are indirectlylabeled with the luminophore. In one embodiment, the labeling moleculedoes not bind to the plurality of target molecules.

In one other aspect, disclosed herein is a composition comprising: aplurality of target molecules, wherein each of the target moleculescomprises a modification moiety and a portion capable of specificbinding to an immobilized probe molecule on a microarray; a particlecomprising a plurality of functional moieties capable of interactingwith the modification moieties of the target molecules; and a bindingmolecule comprising a luminophore, wherein the binding molecule iscapable of specific binding to a subset of the plurality of targetmolecules, and wherein the binding molecule does not bind to theimmobilized probe molecule on the microarray or directly to theparticle, wherein the plurality of target molecules are coupled to theparticle via interaction between the modification moieties and thefunctional moieties, and wherein the plurality of target molecules areindirectly labeled with the luminophore.

In any of the preceding embodiments, the probe molecule can be selectedfrom the group consisting of a polynucleotide, a polypeptide, anantibody, a small molecule compound, a peptide and a carbohydrate. Insome embodiments, the particle is a microparticle. In some aspects, themicroparticle is a paramagnetic microsphere. In some embodiments, themicroparticle has a diameter from about 0.1 micrometers to about 10micrometers. In some embodiments, the particle diameter can be betweenabout 0.1 micrometers and about 0.5 micrometers, about 0.5 micrometersand about 1 micrometer, about 1 micrometer and about 2 micrometers,about 2 micrometers and about 4 micrometers, about 4 micrometers andabout 6 micrometers, about 6 micrometers and about 8 micrometers, orabout 8 micrometers and about 10 micrometers. In some embodiments, theparticle diameter is less than about 0.1 micrometers, or more than about10 micrometers.

In one aspect, disclosed herein is a kit for labeling a target moleculewith a luminophore for detecting the target molecule using a microarray,the kit comprising: a luminophore; means for labeling a subset of aplurality of target molecules with the luminophore; a particlecomprising a plurality of functional moieties, each of the functionalmoieties capable of interacting with a modification moiety of a targetmolecule; and a plurality of probe molecules immobilized on amicroarray, each immobilized probe molecule capable of specific bindingto a target molecule.

In another aspect, disclosed herein is a kit for labeling a targetmolecule with a luminophore for detecting the target molecule using amicroarray, the kit comprising: a luminophore; a particle comprising aplurality of functional moieties, each of the functional moietiescapable of interacting with a modification moiety of a target molecule;means for labeling a subset of the plurality of functional moieties withthe luminophore; and a plurality of probe molecules immobilized on amicroarray, each immobilized probe molecule capable of specific bindingto a target molecule.

In still another aspect, disclosed herein is a kit for labeling a targetmolecule with a luminophore for detecting the target molecule using amicroarray, the kit comprising: a luminophore; a particle comprising aplurality of functional moieties, each of the functional moietiescapable of interacting with a modification moiety of a target molecule;means for introducing the luminophore into or onto the particle; and aplurality of probe molecules immobilized on a microarray, eachimmobilized probe molecule capable of specific binding to a targetmolecule.

In one other aspect, disclosed herein is a kit for labeling a targetmolecule with a luminophore for detecting the target molecule using amicroarray, the kit comprising: a luminophore; a particle comprising aplurality of functional moieties, each of the functional moietiescapable of interacting with a modification moiety of a target molecule;a labeling molecule comprising the modification moiety capable ofinteracting with the functional moiety of the particle; means forlabeling the labeling molecule with the luminophore; and a plurality ofprobe molecules immobilized on a microarray, each immobilized probemolecule capable of specific binding to a target molecule. In oneaspect, the labeling molecule does not bind to the immobilized probemolecules on the microarray.

In yet another aspect, a kit is provided herein for labeling a targetmolecule with a luminophore for detecting the target molecule using amicroarray, the kit comprising: a luminophore; a binding moleculecapable of specific binding to a subset of a plurality of targetmolecules; a particle comprising a plurality of functional moieties,each of the functional moieties capable of interacting with amodification moiety of the target molecules; means for labeling thebinding molecule with the luminophore; and a plurality of probemolecules immobilized on a microarray, each immobilized probe moleculecapable of specific binding to a target molecule. In one embodiment, thebinding molecule does not bind to the immobilized probe molecules on themicroarray or directly to the particle.

In any of the preceding embodiments, the kit can further comprise aprimer comprising a sequence as set forth in Table 2 without the Tagsequence, the biotinylated universal primer sequence at the 5′-terminus,or the Cy3 label, which primer is not a full-length cDNA or afull-length genomic DNA. In any of the preceding embodiments, the kitcan further comprise a primer comprising the sequence as set forth inTable 2.

In any of the preceding embodiments, the kit can further comprise a setof primers for ASPCR amplification of a genetic information comprisingtwo allele-specific primers and a common primer as set forth in Table 2.

In any of the preceding embodiments, the kit can further comprise auniversal tag array comprising at least two of the tag sequences as setforth in Table 1.

In any of the preceding embodiments, the particle can be a microparticleor a paramagnetic microsphere. In any of the preceding embodiments, themicroparticle can have a diameter from about 0.1 micrometers to about 10micrometers.

In any of the preceding embodiments, the functional group can comprise achemical group, a polynucleotide, a polypeptide, an antibody, a smallmolecule compound, a peptide, a carbohydrate, or a combination thereof.In one aspect, the chemical group is an aldehyde, hydroxyl, carboxyl,ester, amine, sulfo, or sulthydryl group. In another aspect, thepolypeptide is streptavidin, neutravidin, or avidin. In yet anotheraspect, the polynucleotide is poly-dT or poly-dA.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic for the layout of a universal tag array formultiplexing, according to one aspect of the present disclosure. FIG. 1Ashows arrangement of the tags as shown in Table 1 on the array. FIG. 1Bshows arrangement of the wild-type probes of polymorphism loci (W) andthe mutant probes of polymorphism loci (M) on the array.

FIG. 2 shows the result of microarray chip scanning, according to amethod depicted in FIG. 3. Hybridization signals are detected in thefirst and third lines corresponding to the wild-type probes.

FIG. 3 is a schematic depicting a method comprising labeling a subset ofa plurality of target molecules with a luminophore, thereby directly orindirectly labeling the plurality of target molecules with theluminophore, according to one aspect of the present disclosure.

FIG. 4 shows the result of microarray chip scanning, according to amethod depicted in FIG. 5. Hybridization signals are detected in thefirst and third lines corresponding to the wild-type probes.

FIG. 5 is a schematic depicting a method comprising labeling a subset ofthe functional moieties of a particle with a luminophore, therebyindirectly labeling the plurality of target molecules with theluminophore, according to one aspect of the present disclosure.

FIG. 6 shows the result of microarray chip scanning, according to amethod depicted in FIG. 7. Hybridization signals are detected in thefirst and third lines corresponding to the wild-type probes.

FIG. 7 is a schematic depicting a method comprising introducing aluminophore into or onto a particle to label the particle, therebyindirectly labeling the plurality of target molecules with theluminophore, according to one aspect of the present disclosure.

FIG. 8 shows the result of microarray chip scanning, according to amethod depicted in FIG. 9. Hybridization signals are detected in thefirst and third lines corresponding to the wild-type probes.

FIG. 9 is a schematic depicting a method comprising providing a labelingmolecule comprising a luminophore and a modification moiety capable ofinteracting with the functional moiety of a particle, thereby indirectlylabeling the plurality of target molecules with the luminophore,according to one aspect of the present disclosure.

FIG. 10 shows the result of microarray chip scanning, according to amethod depicted in FIG. 11. Hybridization signals are detected in thefirst and third lines corresponding to the wild-type probes.

FIG. 11 is a schematic depicting a method comprising providing a bindingmolecule comprising a luminophore and capable of specific binding to asubset of a plurality of target molecules, thereby indirectly labelingthe plurality of target molecules with the luminophore, according to oneaspect of the present disclosure.

DETAILED DESCRIPTION

In some aspects, the present disclosure provides a method that combinesmicroarray-based assays with particles and luminophore-labeled targetmolecules. In some embodiments, the method combines microarray-basedassays with particles, through enriching luminophore-labeled targetnucleic acid fragments, and coupling particles to microarray spotsthrough target-probe hybridization. In some embodiments, the methodcombines microarray-based assays with particles, through enrichingluminophore-labeled double-stranded or single-stranded nucleic acidfragments, and coupling particles to microarray spots throughtarget-probe hybridization. In some aspect, a method disclosed hereinfurther comprises a step of de-multiplexing.

In one aspect, a microarray-based assay is provided, which is used foranalyzing molecular interactions, including interactions betweenpolynucleotides, polypeptides, antibodies, small molecule compounds,peptides and carbohydrates. In certain embodiments, the method disclosedherein comprises labeling a target molecule with a luminophore. In someembodiments, the method further comprises coupling the target moleculeto a particle, and binding to a probe molecule on microarray. Inparticular, multiplexed genetic analysis of nucleic acid fragments canbe implemented. Specific genes, single nucleotide polymorphisms or genemutations, such as deletions, insertions, and indels, can be identified.In one aspect, this technology enables the detection and interpretationof molecular interactions in an efficient way with high sensitivity.

In one aspect, luminophores improve the detection of target-probebinding in microarray-based assays because they exhibit variations insignal intensity or emission spectra resulting from the binding oftarget-probe molecular complex. In some embodiments,luminophore-labeling is integrated with magnetic beads, facilitating theprocess of microarray-based assays. Luminophore-labeled molecules can becoupled to magnetic beads, each of which assembles a large amount ofluminophores at the same time, yielding high intensity of luminescence.High sensitivity detection of molecular interaction is thus achieved.

In some aspects, the present disclosure provides methods, compositions,and kits for improving both sensitivity and specificity ofmicroarray-based assays, concerning with the detection of various SNPsand gene mutations, particularly in clinical settings. Typically,hybridization of labeled nucleic acid targets with surface-immobilizedoligonucleotide probes is the central event in the detection of nucleicacids on microarrays (Riccelli et al., Nucleic Acids Res (2001) 29:996-1004). In some cases, only one of the two strands of DNA products isavailable to hybridize with these probes while the other one competeswith the probes for the targets, acting as a severe interfering factor.Therefore, in some aspects, single-stranded DNA (ssDNA) is enriched, andasymmetric polymerase chain reaction (PCR) is used. In another aspect, aone-step asymmetric PCR without purification process is also used,providing enhanced sensitivity and specificity (Gao et al., Anal Lett(2003) 33: 2849-2863; Zhu et al., supra; Li et al., supra).

In some aspects, microspheres, preferably paramagnetic microspheres areused, due to their easy handling and good biocompatibility, which can befurther improved with the concern of sensitivity (Gao et al., supra).Through capturing double-stranded DNA fragments with microspheres andremoving the unwanted strands by denaturation methods, the yielded ssDNAproducts can be hybridized with microarrays. In one aspect, the purerand more abundance the ssDNA products can be made, the bettersensitivity is expected. As the common symmetric PCR has its propertiesof much higher amplification efficiency and easier design ofmultiplexing compared with asymmetric PCR, in one aspect, the use ofsymmetric PCR and use of ssDNAs can be combined.

Besides ensuring the high sensitivity and specificity, in anotheraspect, combining microarray-based assays with particles andluminophore-labeling facilitates the examination of assay results withappropriate devices. In some aspects, the combination method is used forthe detection of SNP/mutation related to hereditary hearing loss and/orbeta-thalassemia, for multiplexed genetic analysis, or for diagnosis ofclinical samples and disease-associated genetic testing.

In some aspects, the present disclosure provides the followingadvantages:

1. By directly or indirectly labeling the luminophores to the targetmolecules, the present disclosure not only greatly reduces the cost offluorescence detection, but also avoids the PCR inhibition derived fromtraditional fluorescence labeling molecules.

2. The luminophores are labeled to one kind of target molecule (e.g., asubset) in a plurality of modified target molecules, thereby achievingthe goal of reducing the cost of fluorescence labeling of the target,the fluorescence labeling of the primers, and the inhibition of PCRamplification efficiency to a large extent.

3. When the luminophores are introduced into the microparticles, orlabeled to the functional groups that are coated on the surface of themicroparticles, or labeled to a binding molecule, or labeled to alabeling molecule, the method significantly reduces the inhibition ofPCR amplification. In another aspect, the luminophore amount required inthe present disclosure is only about 10% of that required when eachtarget molecule is labeled individually. It should be noted that thelabeling methods disclosed herein can be combined in any suitablemanner, to further increase the labeling efficiency and to reduceinhibition of PCR reactions. For example, luminophores can besimultaneously introduced into the microparticle and labeled to thefunctional groups on the surface of the microparticle, and/or labeled toa binding molecule, and/or labeled to a labeling molecule.

4. When the binding molecule is labeled with a luminophore, the bindingmolecule does not bind to the modification moiety of the modified targetmolecule (therefore does not prevent the target molecule from binding tothe particle). Because the binding molecules can be made short and theydo not bind to the particle, they can efficiently bind to the targetmolecules such that each particle comprises multiple bound targetmolecules that are labeled with the luminophore-labeled bindingmolecules.

A detailed description of one or more embodiments of the claimed subjectmatter is provided below along with accompanying figures that illustratethe principles of the claimed subject matter. The claimed subject matteris described in connection with such embodiments, but is not limited toany particular embodiment. It is to be understood that the claimedsubject matter may be embodied in various forms, and encompassesnumerous alternatives, modifications and equivalents. Therefore,specific details disclosed herein are not to be interpreted as limiting,but rather as a basis for the claims and as a representative basis forteaching one skilled in the art to employ the claimed subject matter invirtually any appropriately detailed system, structure, or manner.Numerous specific details are set forth in the following description inorder to provide a thorough understanding of the present disclosure.These details are provided for the purpose of example and the claimedsubject matter may be practiced according to the claims without some orall of these specific details. It is to be understood that otherembodiments can be used and structural changes can be made withoutdeparting from the scope of the claimed subject matter. It should beunderstood that the various features and functionality described in oneor more of the individual embodiments are not limited in theirapplicability to the particular embodiment with which they aredescribed. They instead can, be applied, alone or in some combination,to one or more of the other embodiments of the disclosure, whether ornot such embodiments are described, and whether or not such features arepresented as being a part of a described embodiment. For the purpose ofclarity, technical material that is known in the technical fieldsrelated to the claimed subject matter has not been described in detailso that the claimed subject matter is not unnecessarily obscured.

Unless defined otherwise, all terms of art, notations and othertechnical and scientific terms or terminology used herein are intendedto have the same meaning as is commonly understood by one of ordinaryskill in the art to which the claimed subject matter pertains. In somecases, terms with commonly understood meanings are defined herein forclarity and/or for ready reference, and the inclusion of suchdefinitions herein should not necessarily be construed to represent asubstantial difference over what is generally understood in the art.Many of the techniques and procedures described or referenced herein arewell understood and commonly employed using conventional methodology bythose skilled in the art.

All publications, including patent documents, scientific articles anddatabases, referred to in this application are incorporated by referencein their entireties for all purposes to the same extent as if eachindividual publication were individually incorporated by reference. If adefinition set forth herein is contrary to or otherwise inconsistentwith a definition set forth in the patents, patent applications,published applications or other publications that are hereinincorporated by reference, the definition set forth herein prevails overthe definition that is incorporated herein by reference. Citation of thepublications or documents is not intended as an admission that any ofthem is pertinent prior art, nor does it constitute any admission as tothe contents or date of these publications or documents.

All headings are for the convenience of the reader and should not beused to limit the meaning of the text that follows the heading, unlessso specified.

The practice of the provided embodiments will employ, unless otherwiseindicated, conventional techniques and descriptions of organicchemistry, polymer technology, molecular biology (including recombinanttechniques), cell biology, biochemistry, and sequencing technology,which are within the skill of those who practice in the art. Suchconventional techniques include polypeptide and protein synthesis andmodification, polynucleotide synthesis and modification, polymer arraysynthesis, hybridization and ligation of polynucleotides, and detectionof hybridization using a label. Specific illustrations of suitabletechniques can be had by reference to the examples herein. However,other equivalent conventional procedures can, of course, also be used.Such conventional techniques and descriptions can be found in standardlaboratory manuals such as Green, et al., Eds., Genome Analysis: ALaboratory Manual Series (Vols. I-IV) (1999); Weiner, Gabriel, Stephens,Eds., Genetic Variation: A Laboratory Manual (2007); Dieffenbach,Dveksler, Eds., PCR Primer: A Laboratory Manual (2003); Bowtell andSambrook, DNA Microarrays: A Molecular Cloning Manual (2003); Mount,Bioinformatics: Sequence and Genome Anazvsis (2004); Sambrook andRussell, Condensed Protocols from Molecular Cloning: A Laboratory Manual(2006); and Sambrook and Russell, Molecular Cloning: A Laboratory Manual(2002) (all from Cold Spring Harbor Laboratory Press); Ausubel et al.eds., Current Protocols in Molecular Biology (1987); T. Brown ed.,Essential Molecular Biology (1991), IRL Press; Goeddel ed., GeneExpression Technology (1991), Academic Press; A. Bothwell et al. eds.,Methods for Cloning and Analysis of Eukaryotic Genes (1990), BartlettPubl.; M. Kriegler, Gene Transfer and Expression (1990), Stockton Press;R. Wu et al. eds., Recombinant DNA Methodology (1989), Academic Press;M. McPherson et al., PCR: A Practical Approach (1991), IRL Press atOxford University Press; Stryer, Biochemistry (4th Ed.) (1995), W. H.Freeman, New York N.Y.; Gait, Oligonucleotide Synthesis: A PracticalApproach (2002), IRL Press, London; Nelson and Cox, Lehninger,Principles of Biochemistry (2000) 3rd Ed., W. H. Freeman Pub., New York,N.Y.; Berg, et al., Biochemistry (2002) 5th Ed., W. H. Freeman Pub., NewYork, N.Y.; D. Weir & C. Blackwell, eds., Handbook of ExperimentalImmunology (1996), Wiley-Blackwell; A. Abbas et al., Cellular andMolecular Immunology (1991, 1994), W.B. Saunders Co.; and J. Coligan etal. eds., Current Protocols in Immunology (1991), all of which areherein incorporated in their entireties by reference for all purposes.

Throughout this disclosure, various aspects of the claimed subjectmatter are presented in a range format. It should be understood that thedescription in range format is merely for convenience and brevity andshould not be construed as an inflexible limitation on the scope of theclaimed subject matter. Accordingly, the description of a range shouldbe considered to have specifically disclosed all the possible sub-rangesas well as individual numerical values within that range. For example,where a range of values is provided, it is understood that eachintervening value, between the upper and lower limit of that range andany other stated or intervening value in that stated range isencompassed within the claimed subject matter. The upper and lowerlimits of these smaller ranges may independently be included in thesmaller ranges, and are also encompassed within the claimed subjectmatter, subject to any specifically excluded limit in the stated range.Where the stated range includes one or both of the limits, rangesexcluding either or both of those included limits are also included inthe claimed subject matter. This applies regardless of the breadth ofthe range.

A. Definitions

As used herein, the singular forms “a”, “an”, and “the” include pluralreferences unless indicated otherwise. For example, “a” dimer includesone or more dimers.

The term “molecule” is used herein to refer to any chemical orbiochemical structure which includes, but is not limited to,polynucleotides, polypeptides, antibodies, small molecule compounds,peptides, and carbohydrates.

A “target” molecule refers to the molecule to be detected by the methodsdescribed in the current disclosure. In the case of a double strandedpolynucleotide, the target molecule may refer to either or both of thecomplementary strands.

The term “luminophore” is used herein to refer to an atom or atomicgrouping in a chemical compound that manifests luminescence.

The term “particle” or “microparticle” is meant to refer to smallparticles, preferred herein in diameter from about 0.01 micrometers toabout 1000 micrometers, for example, from about 0.01 micrometers toabout 0.1 micrometers, from about 0.1 micrometers to about 0.5micrometers, from about 0.5 micrometers to about 1 micrometer, fromabout 1 micrometer to about 5 micrometers, from about 5 micrometers toabout 10 micrometers, from about 10 micrometers to about 100micrometers, from about 100 micrometers to about 500 micrometers, orfrom about 500 micrometers to about 1000 micrometers. In someembodiments, a “particle” or “microparticle” includes an inherentproperty (e.g., magnetization, fluorescence and the like) allowingidentification of each particle or microparticle as belonging to aspecific group. The term “microsphere” is meant to refer to a particle,preferably spherical and usually within the range of from about 0.01micrometers to about 1000 micrometers, for example, from about 0.01micrometers to about 0.1 micrometers, from about 0.1 micrometers toabout 0.5 micrometers, from about 0.5 micrometers to about 1 micrometer,from about 1 micrometer to about 5 micrometers, from about 5 micrometersto about 10 micrometers, from about 10 micrometers to about 100micrometers, from about 100 micrometers to about 500 micrometers, orfrom about 500 micrometers to about 1000 micrometers. In someembodiment, a microsphere may comprise one or more identifying tags(e.g., magnetization, fluorescence and the like) formed together with apolymer, glass, or other matrix, coating or the like. The term “magneticmicrosphere” is meant to refer to a particle within the range of fromabout 0.01 micrometers to about 1000 micrometers (for example, fromabout 0.01 micrometers to about 0.1 micrometers, from about 0.1micrometers to about 0.5 micrometers, from about 0.5 micrometers toabout 1 micrometer, from about 1 micrometer to about 5 micrometers, fromabout 5 micrometers to about 10 micrometers, from about 10 micrometersto about 100 micrometers, from about 100 micrometers to about 500micrometers, or from about 500 micrometers to about 1000 micrometers)including one or more magnetic domains with a polymer, glass, or othermatrix, coating or the like. Neither the term “microsphere” or “magneticmicrosphere” is meant to exclude shapes other than spherical, and suchterms are meant to include other shapes such as globular, flat and thelike.

The term “microarray” is used herein to refer to polynucleotide,polypeptide or chemical microarrays. Specific polynucleotides,polypeptides, antibodies, small molecule compounds, peptides, andcarbohydrates can be immobilized on solid surfaces to form microarrays.

The term “binding” is used herein to refer to an attractive interactionbetween two molecules which results in a stable association in which themolecules are in close proximity to each other. Molecular binding can beclassified into the following types: non-covalent, reversible covalentand irreversible covalent. Molecules that can participate in molecularbinding include polypeptides, polynucleotides, carbohydrates, lipids,and small organic molecules such as pharmaceutical compounds.Polypeptides that form stable complexes with other molecules are oftenreferred to as receptors while their binding partners are calledligands. Polynucleotides can also form stable complex with themselves orothers, for example, DNA-protein complex, DNA-DNA complex, DNA-RNAcomplex.

The term “polypeptide” is used herein to refer to proteins, fragments ofproteins, and peptides, whether isolated from natural sources, producedby recombinant techniques, or chemically synthesized. A polypeptide mayhave one or more modifications, such as a post-translationalmodification (e.g., glycosylation, etc.) or any other modification(e.g., pegylation, etc.). The polypeptide may contain one or morenon-naturally-occurring amino acids (e.g., such as an amino acid with aside chain modification). Polypeptides of the present disclosure maytypically comprise at least about 10 amino acids.

The terms “polynucleotide,” “oligonucleotide,” “nucleic acid” and“nucleic acid molecule” are used interchangeably herein to refer to apolymeric form of nucleotides of any length, and may compriseribonucleotides, deoxyribonucleotides, analogs thereof, or mixturesthereof. This term refers only to the primary structure of the molecule.Thus, the term includes triple-, double- and single-strandeddeoxyribonucleic acid (“DNA”), as well as triple-, double- andsingle-stranded ribonucleic acid (“RNA”). It also includes modified, forexample by alkylation, and/or by capping, and unmodified forms of thepolynucleotide. More particularly, the terms “polynucleotide,”“oligonucleotide,” “nucleic acid” and “nucleic acid molecule” includepolydeoxyribonucleotides (containing 2-deoxy-D-ribose),polyribonucleotides (containing D-ribose), including tRNA, rRNA, hRNA,and mRNA, whether spliced or unspliced, any other type of polynucleotidewhich is an N- or C-glycoside of a purine or pyrimidine base, and otherpolymers containing normucleotidic backbones, for example, polyamide(e.g., peptide nucleic acid (“PNA”)) and polymorpholino (commerciallyavailable from the Anti-Virals, Inc., Corvallis, Oreg., as Neugene)polymers, and other synthetic sequence-specific nucleic acid polymersproviding that the polymers contain nucleobases in a configuration whichallows for base pairing and base stacking, such as is found in DNA andRNA. Thus, these terms include, for example, 3′-deoxy-2′,5′-DNA,oligodeoxyribonucleotide N3′ to P5′ phosphoramidates,2′-O-alkyl-substituted RNA, hybrids between DNA and RNA or between PNAsand DNA or RNA, and also include known types of modifications, forexample, labels, alkylation, “caps,” substitution of one or more of thenucleotides with an analog, intemucleotide modifications such as, forexample, those with uncharged linkages (e.g., methyl phosphonates,phosphotriesters, phosphoramidates, carbamates, etc.), with negativelycharged linkages (e.g., phosphorothioates, phosphorodithioates, etc.),and with positively charged linkages (e.g., aminoalkylphosphoramidates,aminoalkylphosphotriesters), those containing pendant moieties, such as,for example, proteins (including enzymes (e.g. nucleases), toxins,antibodies, signal peptides, poly-L-lysine, etc.), those withintercalators (e.g., acridine, psoralen, etc.), those containingchelates (of, e.g., metals, radioactive metals, boron, oxidative metals,etc.), those containing alkylators, those with modified linkages (e.g.,alpha anomeric nucleic acids, etc.), as well as unmodified forms of thepolynucleotide or oligonucleotide.

It will be appreciated that, as used herein, the terms “nucleoside” and“nucleotide” will include those moieties which contain not only theknown purine and pyrimidine bases, but also other heterocyclic baseswhich have been modified. Such modifications include methylated purinesor pyrimidines, acylated purines or pyrimidines, or other heterocycles.Modified nucleosides or nucleotides can also include modifications onthe sugar moiety, e.g., wherein one or more of the hydroxyl groups arereplaced with halogen, aliphatic groups, or are functionalized asethers, amines, or the like. The term “nucleotidic unit” is intended toencompass nucleosides and nucleotides.

“Nucleic acid probe” and “probe” are used interchangeably and refer to astructure comprising a polynucleotide, as defined above, that contains anucleic acid sequence that can bind to a corresponding target. Thepolynucleotide regions of probes may be composed of DNA, and/or RNA,and/or synthetic nucleotide analogs.

As used herein, “complementary or matched” means that two nucleic acidsequences have at least 50% sequence identity. Preferably, the twonucleic acid sequences have at least 60%, 70%, 80%, 90%, 95%, 96%, 97%,98%, 99% or 100% of sequence identity. “Complementary or matched” alsomeans that two nucleic acid sequences can hybridize under low, middleand/or high stringency condition(s). The percentage of sequence identityor homology is calculated by comparing one to another when aligned tocorresponding portions of the reference sequence.

As used herein, “substantially complementary or substantially matched”means that two nucleic acid sequences have at least 90% sequenceidentity. Preferably, the two nucleic acid sequences have at least 95%,96%, 97%, 98%, 99% or 100% of sequence identity. Alternatively,“substantially complementary or substantially matched” means that twonucleic acid sequences can hybridize under high stringency condition(s).The percentage of sequence identity or homology is calculated bycomparing one to another when aligned to corresponding portions of thereference sequence.

In general, the stability of a hybrid is a function of the ionconcentration and temperature. Typically, a hybridization reaction isperformed under conditions of lower stringency, followed by washes ofvarying, but higher, stringency. Moderately stringent hybridizationrefers to conditions that permit a nucleic acid molecule such as a probeto bind a complementary nucleic acid molecule. The hybridized nucleicacid molecules generally have at least 60% identity, including forexample at least any of 70%, 75%, 80%, 85%, 90%, or 95% identity.Moderately stringent conditions are conditions equivalent tohybridization in 50% formamide, 5×Denhardt's solution, 5×SSPE, 0.2% SDSat 42° C., followed by washing in 0.2×SSPE, 0.2% SDS, at 42° C. Highstringency conditions can be provided, for example, by hybridization in50% formamide, 5×Denhardt's solution, 5×SSPE, 0.2% SDS at 42° C.,followed by washing in 0.1×SSPE, and 0.1% SDS at 65° C. Low stringencyhybridization refers to conditions equivalent to hybridization in 10%formamide, 5×Denhardt's solution, 6×SSPE, 0.2% SDS at 22° C., followedby washing in 1×SSPE, 0.2% SDS, at 37° C. Denhardt's solution contains1% Ficoll, 1% polyvinylpyrolidone, and 1% bovine serum albumin (BSA).20×SSPE (sodium chloride, sodium phosphate, ethylene diamide tetraaceticacid (EDTA)) contains 3M sodium chloride, 0.2M sodium phosphate, and0.025 M EDTA. Other suitable moderate stringency and high stringencyhybridization buffers and conditions are well known to those of skill inthe art and are described, for example, in Sambrook et al., MolecularCloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Press,Plainview, N.Y. (1989); and Ausubel et al., Short Protocols in MolecularBiology, 4th ed., John Wiley & Sons (1999).

Alternatively, substantial complementarity exists when an RNA or DNAstrand will hybridize under selective hybridization conditions to itscomplement. Typically, selective hybridization will occur when there isat least about 65% complementary over a stretch of at least 14 to 25nucleotides, preferably at least about 75%, more preferably at leastabout 90% complementary. See M. Kanehisa Nucleic Acids Res. 12:203(1984).

The terms “homologous”, “substantially homologous”, and “substantialhomology” as used herein denote a sequence of amino acids having atleast 50%, 60%, 70%, 80% or 90% identity wherein one sequence iscompared to a reference sequence of amino acids. The percentage ofsequence identity or homology is calculated by comparing one to anotherwhen aligned to corresponding portions of the reference sequence.

“Multiplexing” or “multiplex assay” herein refers to an assay or otheranalytical method in which the presence of multiple target molecules canbe assayed simultaneously, e.g., by using more than one capture probeconjugate, each of which has at least one different detectioncharacteristic, e.g., fluorescence characteristic (for exampleexcitation wavelength, emission wavelength, emission intensity, FWHM(full width at half maximum peak height), or fluorescence lifetime).

It is understood that aspects and embodiments of the disclosure hereininclude “consisting” and/or “consisting essentially of” aspects andembodiments.

Other objects, advantages and features of the present disclosure willbecome apparent from the following specification taken in conjunctionwith the accompanying drawings.

B. Luminophore

A luminophore is an atom or atomic grouping in a chemical compound thatmanifests luminescence. There exist organic and inorganic luminophores.Luminophores can be divided into two subcategories: fluorophores andphosphors. The difference between luminophores belonging to these twosubcategories is derived from the nature of the excited stateresponsible for the emission of photons. Some luminophores, however,cannot be classified as being exclusively fluorophores or phosphors andexist in the gray area in between. Such cases include transition metalcomplexes (such as ruthenium tris-2,2′-bipyridine) whose luminescencecomes from an excited (nominally triplet) metal-to-ligand chargetransfer (MLCT) state, but which is not a true triplet-state in thestrict sense of the definition. Most luminophores comprise conjugated pisystems or transition metal complexes. There exist purely inorganicluminophores, such as zinc sulfide doped with rare earth metal ions,rare earth metal oxysulfides doped with other rare earth metal ions,yttrium oxide doped with rare earth metal ions, zinc orthosilicate dopedwith manganese ions, etc.

A chromophore is a region in a molecule where the energy differencebetween two different molecular orbitals falls within the range of thevisible spectrum. Visible light that hits the chromophore can thus beabsorbed by exciting an electron from its ground state into an excitedstate. In biological molecules that serve to capture or detect lightenergy, the chromophore is the moiety that causes a conformationalchange of the molecule when hit by light. Chromophores almost alwaysarise in one of two forms: conjugated pi systems and metal complexes. Inthe former, the energy levels that the electrons jump between areextended pi orbitals created by a series of alternating single anddouble bonds, often in aromatic systems. Common examples include retinal(used in the eye to detect light), various food colorings, fabric dyes(azo compounds), lycopene, β-carotene, and anthocyanins. The metalcomplex chromophores arise from the splitting of d-orbitals by bindingof a transition metal to ligands. Examples of such chromophores can beseen in chlorophyll (used by plants for photosynthesis), hemoglobin,hemocyanin, and colorful minerals such as malachite and amethyst.

A fluorophore, in analogy to a chromophore, is a component of a moleculewhich causes a molecule to be fluorescent. It is a functional group in amolecule which will absorb energy of a specific wavelength and re-emitenergy at a different (but equally specific) wavelength. The amount andwavelength of the emitted energy depend on both the fluorophore and thechemical environment of the fluorophore. This technology has particularimportance in the field of biochemistry and protein studies, e.g., inimmunofluorescence and immunohistochemistry. Fluorescein isothiocyanate(FITC), a reactive derivative of fluorescein, has been one of the mostcommon fluorophores chemically attached to other, non-fluorescentmolecules to create new fluorescent molecules for a variety ofapplications. Other common fluorophores include derivatives of rhodamine(TRITC), coumarin, cyanine, the CF Dyes, the FluoProbes, the DyLightFluors, the Oyester(dyes), the Atto dyes, the HiLyte Fluors, and theAlexa Fluors.

These fluorophores can be quantum dots, protein (e.g., green fluorescentprotein (GFP)) or small molecules. Common small molecule dye familiesinclude: xanthene derivatives (fluorescein, rhodamine, Oregon green,eosin, texas red, etc.), cyanine derivatives (cyanine, indocarbocyanine,oxacarbocyanine, thiacarbocyanine, merocyanine, etc.), naphthalenederivatives (dansyl and prodan derivatives), coumarin derivatives,oxadiazole derivatives (pyridyloxazole, nitrobenzoxadiazole,benzoxadiazole, etc.), pyrene derivatives (cascade blue, etc.), BODIPY(Invitrogen), oxazine derivatives (Nile red, Nile blue, cresyl violet,oxazine 170, etc.), acridine derivatives (proflavin, acridine orange,acridine yellow, etc.), arylmethine derivatives (auramine, crystalviolet, malachite green, etc.), CF dye (Biotium), Alexa Fluor(Invitrogen), Atto and Tracy (Sigma), Tetrapyrrole derivatives (porphin,phtalocyanine, bilirubin, etc.), and others (cascade yellow, azure B,acridine orange, DAPI, Hoechst 33258, lucifer yellow, piroxicam, quinineand anthraqinone, squarylium, oligophenylenes, etc.).

In some embodiments, phosphors (or phosphorescent moieties) comprisetransition metal compounds or rare earth compounds of various types. Amaterial can emit light either through incandescence, where all atomsradiate, or by luminescence, where only a small fraction of atoms(called emission centers or luminescence centers) emit light. Ininorganic phosphors, these inhomogeneities in the crystal structure arecreated usually by addition of a trace amount of dopants, impuritiescalled activators. In some cases dislocations or other crystal defectscan play the role of the impurity. The wavelength emitted by theemission center is dependent on the atom itself, and on the surroundingcrystal structure.

The scintillation process in inorganic materials is due to theelectronic band structure found in the crystals. An incoming particlecan excite an electron from the valence band to either the conductionband or the exciton band (located just below the conduction band andseparated from the valence band by an energy gap). This leaves anassociated hole behind, in the valence band. Impurities createelectronic levels in the forbidden gap. The excitons are loosely boundelectron-hole pairs which wander through the crystal lattice until theyare captured as a whole by impurity centers. The latter then rapidlyde-excite by emitting scintillation light (fast component). In case ofinorganic scintillators, the activator impurities are typically chosenso that the emitted light is in the visible range or near-UV wherephotomultipliers are effective. The holes associated with electrons inthe conduction band are independent from the latter. Those holes andelectrons are captured successively by impurity centers exciting certainmetastable states not accessible to the excitons. The delayedde-excitation of those metastable impurity states, slowed down byreliance on the low-probability forbidden mechanism, again results inlight emission (slow component).

C. Microarray

In a high-throughput manner, microarray technologies enable theevaluation of up to tens of thousands of molecular interactionssimultaneously. Microarrays have made significant impact on biology,medicine, drug discovery. DNA microarray-based assays have been widelyused, including the applications for gene expression analysis,genotyping for mutations, single nucleotide polymorphisms (SNPs), andshort tandem repeats (STRs). And polypeptide and chemical microarrayshave emerged as two important tools in the field of proteomics. Chemicalmicroarray, a form of combinatorial libraries, can also be used for leadidentification, as well as optimization of these leads. In this era ofbioterrorism, the development of a microarray capable of detecting amultitude of biological or chemical agents in the environment will be ofgreat interest to the law enforcement agencies.

According to some embodiments of the present disclosure, assay methodsfor analysis of molecular interactions are provided. According to someembodiments of the present disclosure, assay methods for multiplexedanalysis of target polynucleotides are provided. The inventivetechnology improves specificity and sensitivity of microarray-basedassays while reducing the cost of performing genetic assays.

The target molecules include polynucleotides, polypeptides, antibodies,small molecule compounds, peptides, and carbohydrates.

As those of ordinary skill in the art will recognize, the presentdisclosure has an enormous number of applications, especially in assaysand techniques for pharmaceutical development and diagnostics. Theassays may be designed, for example, to detect polynucleotide moleculesassociated with any of a number of infectious or pathogenic agentsincluding fungi, bacteria, mycoplasma, rickettsia, chlamydia, viruses,and protozoa, or to detect polynucleotide fragments associated withsexually transmitted disease, pulmonary disorders, gastrointestinaldisorders, cardiovascular disorders, etc.

A microarray is a multiplex technology widely used in molecular biologyand medicine. Microarrays can be fabricated using a variety oftechnologies, including printing with fine-pointed pins,photolithography using pre-made masks, photolithography using dynamicmicromirror devices, ink-jet printing, microcontact printing, orelectrochemistry on microelectrode arrays. In standard microarrays, theprobe molecules are attached via surface engineering to a solid surfaceof supporting materials, which include glass, silicon, plastic,hydrogels, agaroses, nitrocellulose and nylon.

The microarray results for the detection of fluorescence-labeled targetmolecules can be viewed with a suitable method, e.g., by a CCD in brightfield (left panel), under a fluorescence microscopy (middle panel), andby a commercial fluorescence microarray scanner with pseudo-colorprocessing (right panel).

For DNA microarray, it comprises an arrayed series of microscopic spotsof DNA oligonucleotides, known as probes. This can be a short section ofa gene or other DNA element that are used to hybridize a complementarypolynucleotide sample (called target) under stringent conditions.Targets in solution are usually detected and quantified by detection offluorophore-, silver-, or chemiluminescence-labeled targets hybridizedon microarray. Since an array can contain several to tens of thousandsof probes, a microarray experiment can accomplish many genetic tests inparallel.

The systems described herein may comprise two or more probes that detectthe same target polynucleotide. For example, in some embodiments wherethe system is a microarray, the probes may be present in multiple (suchas any of 2, 3, 4, 5, 6, 7, or more) copies on the microarray. In someembodiments, the system comprises different probes that detect the sametarget polynucleotide. For example, these probes may bind to different(overlapping or non-overlapping) regions of the target polynucleotide.

Any probes that are capable of determining the levels of targetpolynucleotide can be used. In some embodiments, the probe may be anoligonucleotide. It is understood that, for detection of targetpolynucleotides, certain sequence variations are acceptable. Thus, thesequence of the oligonucleotides (or their complementary sequences) maybe slightly different from those of the target polynucleotides describedherein. Such sequence variations are understood by those of ordinaryskill in the art to be variations in the sequence that do notsignificantly affect the ability of the oligonucleotide to determinetarget polynucleotide levels. For example, homologs and variants ofthese oligonucleotide molecules possess a relatively high degree ofsequence identity when aligned using standard methods. Oligonucleotidesequences encompassed by the present disclosure have at least 40%,including for example at least about any of 50%, 60%, 70%, 80%, 90%,95%, 99%, or 100% sequence identity to the sequence of the targetpolynucleotides described herein. In some embodiments, theoligonucleotide comprises a portion for detecting the targetpolynucleotides and another portion. Such other portion may be used, forexample, for attaching the oligonucleotides to a substrate. In someembodiments, the other portion comprises a non-specific sequence (suchas poly-T or poly-dT) for increasing the distance between thecomplementary sequence portion and the surface of the substrate.

The oligonucleotides for the systems described herein include, forexample, DNA, RNA, PNA, ZNA, LNA, combinations thereof, and/or modifiedforms thereof. They may also include a modified oligonucleotidebackbone. In some embodiments, the oligonucleotide comprises at leastabout any of 5, 6, 7, 8, 9, 10, 12, 13, 14, 15, 16, 17, 18, 19, 20, ormore continuous oligonucleotides complementary or identical to all orpart of target polynucleotides described herein. A singleoligonucleotide may comprise two or more such complementary sequences.In some embodiments, there may be a reactive group (such as an amine)attached to the 5′ or 3′ end of the oligonucleotide for attaching theoligonucleotide to a substrate.

In some embodiments, the probes are oligonucleotides. Oligonucleotidesforming the array may be attached to the substrate by any number of waysincluding, but not limiting to, (i) in situ synthesis (e.g.,high-density oligonucleotide arrays) using photolithographic techniques;(ii) spotting/printing at medium to low density on glass, silicon, nylonor nitrocellulose; (iii) masking; and (iv) dot-blotting on a nylon ornitrocellulose hybridization membrane. Oligonucleotides may also benon-covalently immobilized on the substrate by binding to anchors in afluid phase such as in microtiter wells, microchannels or capillaries.

Several techniques are well-known in the art for attachingpolynucleotides to a solid substrate such as a glass slide. One methodis to incorporate modified bases or analogs that contain a moiety thatis capable of attachment to a solid substrate, such as an amine group, aderivative of an amine group or another group with a positive charge,into the amplified polynucleotides. The amplified product is thencontacted with a solid substrate, such as a glass slide, which may becoated with an aldehyde or another reactive group which can form acovalent link with the reactive group that is on the amplified productand become covalently attached to the glass slide. Microarrayscomprising the amplified products can be fabricated using a Biodot(BioDot, Inc. Irvine, Calif.) spotting apparatus and aldehyde-coatedglass slides (CEL Associates, Houston, Tex.). Amplification products canbe spotted onto the aldehyde-coated slides, and processed according topublished procedures (Schena et al., Proc. Natl. Acad. Sci. U.S.A.(1995), 93:10614-10619). Arrays can also be printed by robotics ontoglass, nylon (Ramsay, G., Nature Biotechnol. (1998), 16:40-44),polypropylene (Matson, et al., Anal Biochem. (1995), 224(1):110-6), andsilicone slides (Marshall and Hodgson, Nature Biotechnol. (1998),16:27-31). Other approaches to array assembly include finemicropipetting within electric fields (Marshall, and Hodgson, NatureBiotechnol. (1998), 16:27-31), and spotting the polynucleotides directlyonto positively coated plates. Methods such as those using amino propylsilicon surface chemistry are also known in the art, as disclosed atcmgm.stanford.edu/pbrown/.

The assays of the present disclosure may be implemented in a multiplexformat. Multiplex methods are provided employing 2, 3, 4, 5, 10, 15, 20,25, 50, 100, 200, 500, 1000 or more different capture probes which canbe used simultaneously to assay for amplification products fromcorresponding different target polynucleotides. In some embodiments,multiplex methods can also be used to assay for polynucleotide targetsequences which have not undergone an amplification procedure. Methodsamenable to multiplexing, such as those taught herein, allow acquisitionof greater amounts of information from smaller specimens. The need forsmaller specimens increases the ability of an investigator to obtainsamples from a larger number of individuals in a population to validatea new assay or simply to acquire data, as less invasive techniques areneeded.

Where different substrates are included in a multiplex assay as part ofthe capture probe conjugates, the different substrates can be encoded sothat they can be distinguished. Any encoding scheme can be used;conveniently, the encoding scheme can employ one or more differentfluorophores, which can be fluorescent semiconductor nanocrystals. Highdensity spectral coding schemes can be used.

One or more different populations of spectrally encoded capture probeconjugates can be created, each population comprising one or moredifferent capture probes attached to a substrate comprising a known ordeterminable spectral code comprising one or more semiconductornanocrystals or fluorescent nanoparticle. Different populations of theconjugates, and thus different assays, can be blended together, and theassay can be performed in the presence of the blended populations. Theindividual conjugates are scanned for their spectral properties, whichallows the spectral code to be decoded and thus identifies thesubstrate, and therefore the capture probe(s) to which it is attached.Because of the large number of different semiconductor nanocrystals andfluorescent nanoparticles and combinations thereof which can bedistinguished, large numbers of different capture probes andamplification products can be simultaneously interrogated.

D. Particles

The present disclosure provides particles, microparticles or beads,preferably magnetic beads, to be used for the microarray-based assay.Particles or beads can be prepared from a variety of different polymers,including but not limited to polystyrene, cross-linked polystyrene,polyacrylic acid, polylactic acid, polyglycolic acid, poly(lactidecoglycolide), polyanhydrides, poly(methyl methacrylate),poly(ethylene-co-vinyl acetate), polysiloxanes, polymeric silica,latexes, dextran polymers and epoxies. The materials have a variety ofdifferent properties with regard to swelling and porosity, which arewell understood in the art. Preferably, the beads are in the size rangeof approximately 10 nanometers to 1 millimeter, preferably 100nanometers to 10 micrometers, and can be manipulated using normalsolution techniques when suspended in a solution. The terms “particle,”“bead,” “sphere,” “microparticle,” “microbead” and “microsphere” areused interchangeably herein.

The suitable chemical compositions for the magnetic particles may beferromagnetic materials and include rare earth containing materials suchas, e.g., iron-cobalt, iron-platinum, samarium-cobalt,neodynium-iron-boride, and the like. Other magnetic materials, e.g.,superparamagnetic materials such as iron oxides (Fe₃O₄) may be used aswell. Among the preferred magnetic materials are included iron-cobalt assuch material is generally easier to magnetize, has a strongermagnetization (about 1.7 Tesla) and is less susceptible to corrosion.

Because of the use of particles, expensive readout devices for resultsmay not be necessary. Particles on the microarray spots can be vieweddirectly with naked eyes if the sizes in diameters of these spots arelarger than 0.03 millimeters. In another way, assay results with anyspot sizes, from 0.01 millimeters to 5 millimeters in diameter, can bephotographed with an ordinary camera or viewed under an appropriatemagnification microscope.

E. Target Polynucleotide

The target polynucleotide can be single-stranded, double-stranded, orhigher order, and can be linear or circular. Exemplary single-strandedtarget polynucleotides include mRNA, rRNA, tRNA, hnRNA, microRNA, ssRNAor ssDNA viral genomes and viroids, although these polynucleotides maycontain internally complementary sequences and significant secondarystructure. Exemplary double-stranded target polynucleotides includegenomic DNA, mitochondrial DNA, chloroplast DNA, dsRNA or dsDNA viralgenomes, plasmids, phages, shRNA (a small hairpin RNA or short hairpinRNA), and siRNA (small/short interfering RNA). The target polynucleotidecan be prepared recombinantly, synthetically or purified from abiological source. The target polynucleotide may be purified to removeor diminish one or more undesired components of the sample or toconcentrate the target polynucleotide prior to amplification.Conversely, where the target polynucleotide is too concentrated for aparticular assay, the target polynucleotide may first be diluted.

Following sample collection and optional nucleic acid extraction andpurification, the nucleic acid portion of the sample comprising thetarget polynucleotide can be subjected to one or more preparativetreatments. These preparative treatments can include in vitrotranscription (IVT), labeling, fragmentation, amplification and otherreactions. mRNA can first be treated with reverse transcriptase and aprimer, which can be the first primer comprising the targetnon-complementary region, to create cDNA prior to detection and/orfurther amplification; this can be done in vitro with extracted orpurified mRNA or in situ, e.g., in cells or tissues affixed to a slide.Nucleic acid amplification increases the copy number of sequences ofinterest and can be used to incorporate a label into an amplificationproduct produced from the target polynucleotide using a labeled primeror labeled nucleotide. A variety of amplification methods are suitablefor use, including the polymerase chain reaction method (PCR),transcription mediated amplification (TMA), the ligase chain reaction(LCR), self sustained sequence replication (3SR), nucleic acidsequence-based amplification (NASBA), rolling circle amplification(RCA), loop-mediated isothermal amplification (LAMP), the use of Q Betareplicase, reverse transcription, nick translation, and the like,particularly where a labeled amplification product can be produced andutilized in the methods taught herein.

Any nucleotides may be detected by the present devices and methods.Examples of such nucleotides include AMP, GMP, CMP, UMP, ADP, GDP, CDP,UDP, ATP, GTP, CTP, UTP, dAMP, dGMP, dCMP, dTMP, dADP, dGDP, dCDP, dTDP,dATP, dGTP, dCTP and dTTP.

In some embodiments, the target polynucleotide does not have a labeldirectly incorporated in the sequence. When the target polynucleotide ismade with a directly incorporated label or so that a label can bedirectly bound to the target polynucleotide, this label is one whichdoes not interfere with detection of the capture probe conjugatesubstrate and/or the report moiety label.

Where the target polynucleotide is single-stranded, the first cycle ofamplification forms a primer extension product complementary to thetarget polynucleotide. If the target polynucleotide is single-strandedRNA, a reverse transcriptase is used in the first amplification toreverse transcribe the RNA to DNA, and additional amplification cyclescan be performed to copy the primer extension products. The primers fora PCR must, of course, be designed to hybridize to regions in theircorresponding template that will produce an amplifiable segment; thus,each primer must hybridize so that its 3′ nucleotide is base-paired witha nucleotide in its corresponding template strand that is located 3′from the 3′ nucleotide of the primer used to prime the synthesis of thecomplementary template strand.

The target polynucleotide may be amplified by contacting one or morestrands of the target polynucleotide with a primer and a polymerasehaving suitable activity to extend the primer and copy the targetpolynucleotide to produce a full-length complementary polynucleotide ora smaller portion thereof. Any enzyme having a polymerase activity whichcan copy the target polynucleotide can be used, including DNApolymerases, RNA polymerases, reverse transcriptases, enzymes havingmore than one type of polymerase activity. The polymerase can bethermolabile or thermostable. Mixtures of enzymes can also be used.Exemplary enzymes include: DNA polymerases such as DNA Polymerase I(“Pol I”), the Klenow fragment of Pol I, T4, T7, Sequenase™ T7,Sequenase™ Version 2.0 T7, Tub, Taq, Tth, Pfx, Pfu, Tsp, Tfl, Tli andPyrococcus sp GB-D DNA polymerases; RNA polymerases such as E. coli,SP6, T3 and T7 RNA polymerases; and reverse transcriptases such as AMV,M-MuLV, MMLV, RNAse H minus MMLV (SuperScript™), SuperScript™ II,ThermoScript™, HIV-1, and RAV2 reverse transcriptases. All of theseenzymes are commercially available. Exemplary polymerases with multiplespecificities include RAV2 and Tli (exo-) polymerases. Exemplarythermostable polymerases include Tub, Taq, Tth, Pfx, Pfu, Tsp, Tfl, Tliand Pyrococcus sp. GB-D DNA polymerases.

Suitable reaction conditions are chosen to permit amplification of thetarget polynucleotide, including pH, buffer, ionic strength, presenceand concentration of one or more salts, presence and concentration ofreactants and cofactors such as nucleotides and magnesium and/or othermetal ions, optional co-solvents, temperature, thermal cycling profilefor amplification schemes comprising a polymerase chain reaction, andmay depend in part on the polymerase being used as well as the nature ofthe sample. Co-solvents include formamide (typically at from about 2 toabout 10%), glycerol (typically at from about 5 to about 10%), and DMSO(typically at from about 0.9 to about 10%). Techniques may be used inthe amplification scheme in order to minimize the production of falsepositives or artifacts produced during amplification. These include“touchdown” PCR, hot-start techniques, use of nested primers, ordesigning PCR primers so that they form stem-loop structures in theevent of primer-dimer formation and thus are not amplified. Techniquesto accelerate PCR can be used, for example centrifugal PCR, which allowsfor greater convection within the sample, and comprising infraredheating steps for rapid heating and cooling of the sample. One or morecycles of amplification can be performed. An excess of one primer can beused to produce an excess of one primer extension product during PCR;preferably, the primer extension product produced in excess is theamplification product to be detected. A plurality of different primersmay be used to amplify different regions of a particular polynucleotidewithin the sample. Where the amplification reaction comprises multiplecycles of amplification with a polymerase, as in PCR, it is desirable todissociate the primer extension product(s) formed in a given cycle fromtheir template(s). The reaction conditions are therefore altered betweencycles to favor such dissociation; typically this is done by elevatingthe temperature of the reaction mixture, but other reaction conditionscan be altered to favor dissociation, for example lowering the saltconcentration and/or raising the pH of the solution in which thedouble-stranded polynucleotide is dissolved. Although it is preferableto perform the dissociation in the amplification reaction mixture, thepolynucleotides may be first isolated using any effective technique andtransferred to a different solution for dissociation, then reintroducedinto an amplification reaction mixture for additional amplificationcycles.

In some aspects, the assay disclosed herein can be multiplexed, e.g.,multiple distinct assays can be run simultaneously, by using differentpairs of primers directed at different targets, which can be unrelatedtargets, or different alleles or subgroups of alleles from, orchromosomal rearrangements at, the same locus. This allows thequantitation of the presence of multiple target polynucleotides in asample (e.g., specific genes in a cDNA library). All that is required isan ability to uniquely identify the different second polynucleotideextension products in such an assay, through either a unique capturesequence or a unique label.

Amplified target polynucleotides may be subjected to post-amplificationtreatments. For example, in some cases, it may be desirable to fragmentthe amplification products prior to hybridization with a polynucleotidearray, in order to provide segments which are more readily accessibleand which avoid looping and/or hybridization to multiple capture probes.Fragmentation of the polynucleotides can be carried out by any methodproducing fragments of a size useful in the assay being performed;suitable physical, chemical and enzymatic methods are known in the art.

Amplified target polynucleotides may also be coupled to the particles,either directly or through modifications to the polynucleotides and/orthe particles. In some embodiments, the target polynucleotides aremodified, such as biotinylation. In some other embodiments, theparticles are modified with a functional group, such as streptavidin,neutravidin, avidin, etc. The target polynucleotides may be coupled tothe particles through such modifications and functional groups. Fordouble stranded polynucleotides, following the coupling of the targetpolynucleotides to the particles, single-stranded target polynucleotidescan be prepared by denaturation methods by a chemical reaction, enzymeor heating, or a combination thereof, while coupled to the particles. Insome embodiments, the chemical reaction uses urea, formamide, methanol,ethanol, an enzyme, or NaOH. In some embodiments, enzymatic methodsinclude exonuclease and Uracil-N-glycosylase. In some other embodiments,the double-stranded target polynucleotide is heat denatured at anappropriate temperature from about 30° C. to about 95° C.

The method of the present disclosure is suitable for use in ahomogeneous multiplex analysis of multiple target polynucleotides in asample. Multiple target polynucleotides can be generated byamplification of a sample by multiple amplification oligonucleotideprimers or sets of primers, each primer or set of primers specific foramplifying a particular polynucleotide target sequence. For example, asample can be analyzed for the presence of multiple viral polynucleotidetarget sequences by amplification with primers specific foramplification of each of multiple viral target sequences, including,e.g., human immunodeficiency virus (HIV), hepatitis B virus (HBV),hepatitis C virus (HCV), hepatitis A virus (HAV), parvovirus B 19, WestNile Virus, hantavirus, severe acute respiratory syndrome-associatedcoronavirus (SARS), etc.

The portion of the sample comprising or suspected of comprising thetarget polynucleotide can be any source of biological material whichcomprises polynucleotides that can be obtained from a living organismdirectly or indirectly, including cells, tissue or fluid, and thedeposits left by that organism, including viruses, mycoplasma, andfossils. The sample can also comprise a target polynucleotide preparedthrough synthetic means, in whole or in part. Typically, the sample isobtained as or dispersed in a predominantly aqueous medium. Non-limitingexamples of the sample include blood, plasma, urine, semen, milk,sputum, mucus, a buccal swab, a vaginal swab, a rectal swab, anaspirate, a needle biopsy, a section of tissue obtained for example bysurgery or autopsy, plasma, serum, spinal fluid, lymph fluid, theexternal secretions of the skin, respiratory, intestinal, andgenitourinary tracts, tears, saliva, tumors, organs, samples of in vitrocell culture constituents (including but not limited to conditionedmedium resulting from the growth of cells in cell culture medium,putatively virally infected cells, recombinant cells, and cellcomponents), and a recombinant source, e.g., a library, comprisingpolynucleotide sequences.

The sample can be a positive control sample which is known to containthe target polynucleotide or a surrogate thereof. A negative controlsample can also be used which, although not expected to contain thetarget polynucleotide, is suspected of containing it, and is tested inorder to confirm the lack of contamination by the target polynucleotideof the reagents used in a given assay, as well as to determine whether agiven set of assay conditions produces false positives (a positivesignal even in the absence of target polynucleotide in the sample).

The sample can be diluted, dissolved, suspended, extracted or otherwisetreated to solubilize and/or purify any target polynucleotide present orto render it accessible to reagents which are used in an amplificationscheme or to detection reagents. Where the sample contains cells, thecells can be lysed or permeabilized to release the polynucleotideswithin the cells. Permeabilization buffers can be used to lyse cellswhich allow further steps to be performed directly after lysis, forexample a polymerase chain reaction.

F. Genetic Information

Any kind of genetic information can be the subject of the presentlyclaimed method of microarray based analysis. For example, the geneticinformation may be a mutation selected from the group consisting of asubstitution, an insertion, a deletion and an indel. In one embodiment,the genetic information is a single nucleotide polymorphism (SNP). Inone embodiment, the genetic information is a gene. In one embodiment,the genetic information is a genetic product including a polypeptide, anantibody, a small molecule compound, a peptide and a carbohydrate. Inanother embodiment, the genetic information is associated with a diseasecaused by an infectious or pathogenic agent selected from the groupconsisting of a fungus, a bacterium, a mycoplasma, a rickettsia, achlamydia, a virus and a protozoa. In yet another embodiment, thegenetic information is associated with a sexually transmitted disease,cancer, cerebrovascular disease, heart disease, respiratory disease,coronary heart disease, diabetes, hypertension, Alzheimer's disease,neurodegenerative disease, chronic obstructive pulmonary disease,autoimmune disease, cystic fibrosis, spinal muscular atrophy, betathalassemia, phenylalanine hydroxylase deficiency, Duchenne musculardystrophy, or hereditary hearing loss. In still another embodiment, thegenetic information is associated with hereditary hearing loss.

The allele of the target gene may be caused by single base substitution,insertion, or deletion, or by multiple-base substitution, insertion ordeletion, or indel. Furthermore, modifications to nucleotidic unitsinclude rearranging, appending, substituting for or otherwise alteringfunctional groups on the purine or pyrimidine base which form hydrogenbonds to a respective complementary pyrimidine or purine. The resultantmodified nucleotidic unit optionally may form a base pair with othersuch modified nucleotidic units but not with A, T, C, G or U. Basicsites may be incorporated which do not prevent the function of thepolynucleotide. Some or all of the residues in the polynucleotide canoptionally be modified in one or more ways.

Standard A-T and G-C base pairs form under conditions which allow theformation of hydrogen bonds between the N3-H and C4-oxy of thymidine andthe N1 and C6-NH2, respectively, of adenosine and between the C2-oxy, N3and C4-NH2, of cytidine and the C2-NH2, N—H and C6-oxy, respectively, ofguanosine. Thus, for example, guanosine(2-amino-6-oxy-9-O-D-ribofuran-osyl-purine) may be modified to formisoguanosine (2-oxy-6-amino-9-β-D-ribofuranosyl-purine). Suchmodification results in a nucleoside base which will no longereffectively form a standard base pair with cytosine. However,modification of cytosine (1-β-D-ribofuranosyl-2-oxy-4-amino-pyrimidine)to form isocytosine (1-β-D-ribofuranosyl-2-amino-4-oxy-pyrimidine)results in a modified nucleotide which will not effectively base pairwith guanosine but will form a base pair with isoguanosine (U.S. Pat.No. 5,681,702). Isocytosine is available from Sigma Chemical Co. (St.Louis, Mo.); isocytidine may be prepared by the method described bySwitzer et al. (1993) Biochemistry 32:10489-10496 and references citedtherein; 2′-deoxy-5-methyl-isocytidine may be prepared by the method ofTor et al. (1993) J. Am. Chem. Soc. 115:4461-4467 and references citedtherein; and isoguanine nucleotides may be prepared using the methoddescribed by Switzer et al. (1993), supra, and Mantsch et al. (1993)Biochem. 14:5593-5601, or by the method described in U.S. Pat. No.5,780,610. Other normatural base pairs may be synthesized by the methoddescribed in Piccirilli et al. (1990) Nature 343:33-37 for the synthesisof 2,6-diaminopyrimidine and its complement(1-methylpyrazolo-[4,3]pyrimidine-5,7-(4H,6H)-dione). Other suchmodified nucleotidic units which form unique base pairs are known, suchas those described in Leach et al. (1992) J. Am. Chem. Soc.114:3675-3683 and Switzer et al., supra.

A polymorphic region as defined herein is a portion of a genetic locusthat is characterized by at least one polymorphic site. A genetic locusis a location on a chromosome which is associated with a gene, aphysical feature, or a phenotypic trait. A polymorphic site is aposition within a genetic locus at which at least two alternativesequences have been observed in a population. A polymorphic region asdefined herein is said to “correspond to” a polymorphic site, that is,the region may be adjacent to the polymorphic site on the 5′ side of thesite or on the 3′ side of the site, or alternatively may contain thepolymorphic site. A polymorphic region includes both the sense andantisense strands of the polynucleotide comprising the polymorphic site,and may have a length of from about 100 to about 5000 base pairs. Forexample, a polymorphic region may be all or a portion of a regulatoryregion such as a promoter, 5′ UTR, 3′ UTR, an intron, an exon, or thelike. A polymorphic or allelic variant is a genomic DNA, cDNA, mRNA orpolypeptide having a nucleotide or amino acid sequence that comprises apolymorphism. A polymorphism is a sequence variation observed at apolymorphic site, including nucleotide substitutions (single nucleotidepolymorphisms or SNPs), insertions, deletions, indels andmicrosatellites. Polymorphisms may or may not result in detectabledifferences in gene expression, protein structure, or protein function.Preferably, a polymorphic region of the present disclosure has a lengthof about 1000 base pairs. More preferably, a polymorphic region of thepresent disclosure has a length of about 500 base pairs. Mostpreferably, a polymorphic region of the present disclosure has a lengthof about 200 base pairs.

A haplotype as defined herein is a representation of the combination ofpolymorphic variants in a defined region within a genetic locus on oneof the chromosomes in a chromosome pair. A genotype as used herein is arepresentation of the polymorphic variants present at a polymorphicsite.

Those of ordinary skill will recognize that oligonucleotidescomplementary to the polymorphic regions described herein must becapable of hybridizing to the polymorphic regions under conditions ofstringency such as those employed in primer extension-based sequencedetermination methods, restriction site analysis, nucleic acidamplification methods, ligase-based sequencing methods, mismatch-basedsequence determination methods, microarray-based sequence determinationmethods, and the like.

Congenital hearing loss affects one in 1,000 live births andapproximately 50% of these cases are hereditary. Among Chinese disabledpersons, hearing loss population is the second largest. SNPs/mutationsin GJB2, SLC26A4 and 12S rRNA are the prevalent causes of inheritedhearing loss. In one aspect, the present disclosure meets the need ofSNP/mutation detection from various deafness patients or even healthypersons, which also serves as an example to support the applicability ofa method disclosed herein.

G. Oligonucleotide Primers for Amplification of Target Polynucleotides

In certain aspects, the present disclosure is also embodied inoligonucleotide primer pairs suitable for use in the polymerase chainreaction (PCR) or in other nucleic acid amplification methods. Those ofordinary skill will be able to design suitable oligonucleotide primerpairs using knowledge readily available in the art, in combination withthe teachings herein. Specific oligonucleotide primer pairs of thisembodiment include the oligonucleotide primer pairs set forth in Table2, which are suitable for amplifying the polymorphic regionscorresponding to polymorphic sites in GJB2, SLC26A4 and 12S rRNA. Thoseof ordinary skill will recognize that other oligonucleotide primer pairssuitable for amplifying the polymorphic regions in GJB2, SLC26A4 and 12SrRNA can be designed without undue experimentation.

In some variations a SNP/mutation corresponds to at least twoallele-specific primers. One allele-specific primer comprises a sequenceidentical or complementary to a region of the wild-type allele of atarget fragment containing the SNP/mutation locus. Each of the otherallele-specific primers comprises a sequence identical or complementaryto a region of the mutant allele of a target fragment containing theSNP/mutation locus. The allele-specific primers may terminate at their3′ ends at the SNP/mutation locus. To increase the capability ofdifferentiation between the wild-type and mutant alleles of targetgenes, an artificial mismatch in the allele-specific primers may beintroduced. The artificial mismatch can be a natural base or anucleotide analog. Each of the PCR primer pairs of the presentdisclosure may be used in any PCR method. For example, a PCR primer pairof the present disclosure may be used in the methods disclosed in U.S.Pat. Nos. 4,683,195; 4,683,202, 4,965,188; 5,656,493; 5,998,143;6,140,054; WO 01/27327; WO 01/27329; and the like. The PCR primer pairsof the present disclosure may also be used in any of the commerciallyavailable machines that perform PCR, such as any of the GeneAmp® Systemsavailable from Applied Biosystems.

The present primers can comprise any suitable types of nucleic acids,e.g., DNA, RNA, PNA or a derivative thereof. Preferably, the primerscomprise a nucleotide sequence, or a complementary strand thereof, thatis set forth in Table 2. Also preferably, the primers are labeled, e.g.,a chemical, an enzymatic, an immunogenic, a radioactive, a fluorescent,a luminescent and a FRET label.

The oligonucleotide primers can be produced by any suitable method. Forexample, the primers can be chemically synthesized (See generally,Ausubel (Ed.) Current Protocols in Molecular Biology, 2.11. Synthesisand purification of oligonucleotides, John Wiley & Sons, Inc. (2000)),isolated from a natural source, produced by recombinant methods or acombination thereof. Synthetic oligonucleotides can also be prepared byusing the triester method of Matteucci et al., J. Am. Chem. Soc.,3:3185-3191 (1981). Alternatively, automated synthesis may be preferred,for example, on an Applied Biosynthesis DNA synthesizer using cyanoethylphosphoramidite chemistry. Preferably, the primers are chemicallysynthesized.

Suitable bases for preparing the oligonucleotide primers of the presentdisclosure may be selected from naturally occurring nucleotide basessuch as adenine, cytosine, guanine, uracil, and thymine. It may also beselected from nonnaturally occurring or “synthetic” nucleotide basessuch as 8-oxo-guanine, 6-mercaptoguanine, 4-acetylcytidine,5-(carboxyhydroxyethyl) uridine, 2′-O-methylcytidine,5-carboxymethylamino-methyl-2-thioridine, 5-carboxymethylaminomethyluridine, dihydrouridine, 2′-O-methylpseudouridine,beta-D-galactosylqueosine, 2′-Omethylguanosine, inosine,N6-isopentenyladenosine, 1-methyladenosine, 1-methylpseudouridine,1-methylguanosine, 1-methylinosine, 2,2-dimethylguanosine,2-methyladenosine, 2-methylguanosine, 3-methylcytidine,5-methylcytidine, N6-methyladenosine, 7-methylguanosine,5-methylaminomethyluridine, 5-methoxyaminomethyl-2-thiouridine,beta-D-mannosylqueosine, 5-methoxycarbonylmethyluridine,5-methoxyuridine, 2-methylthio-N6-isopentenyladenosine,N-((9-.beta.-D-ribofuranosyl-2-methylthiopurine-6-yl)carbamoyl)threonine,N-((9-beta-D-ribofuranosylpurine-6-yl)N-methylcarbamoyl) threonine,uridine-5-oxyacetic acid methylester, uridine-5-oxyacetic acid,wybutoxosine, pseudouridine, queosine, 2-thiocytidine,5-methyl-2-thiouridine, 2-thiouridine, 2-thiouridine, 5-methyluridine,N-((9-beta-D-ribofuranosylpurine-6-yl) carbamoyl) threonine,2′-O-methyl-5-methyluridine, 2′-O-methyluridine, wybutosine, and3-(3-amino-3-carboxypropyl) uridine.

Likewise, chemical analogs of oligonucleotides (e.g., oligonucleotidesin which the phosphodiester bonds have been modified, e.g., to themethylphosphonate, the phosphotriester, the phosphorothioate, thephosphorodithioate, or the phosphoramidate) may also be employed.Protection from degradation can be achieved by use of a “3′-end cap”strategy by which nuclease-resistant linkages are substituted forphosphodiester linkages at the 3′ end of the oligonucleotide (Shaw etal., Nucleic Acids Res., 19:747 (1991)). Phosphoramidates,phosphorothioates, and methylphosphonate linkages all functionadequately in this manner. More extensive modification of thephosphodiester backbone has been shown to impart stability and may allowfor enhanced affinity and increased cellular permeation ofoligonucleotides (Milligan et al., J. Med. Chem., 36:1923 (1993)). Manydifferent chemical strategies have been employed to replace the entirephosphodiester backbone with novel linkages. Backbone analogues includephosphorothioate, phosphorodithioate, methylphosphonate,phosphoramidate, boranophosphate, phosphotriester, formacetal,3′-thioformacetal, 5′-thioformacetal, 5′-thioether, carbonate,5′-N-carbamate, sulfate, sulfonate, sulfamate, sulfonamide, sulfone,sulfite, sulfoxide, sulfide, hydroxylamine, methylene (methylimino)(MMI) or methyleneoxy (methylimino) (MOMI) linkages. Phosphorothioateand methylphosphonate-modified oligonucleotides are particularlypreferred due to their availability through automated oligonucleotidesynthesis. The oligonucleotide may be a “peptide nucleic acid” such asdescribed by (Milligan et al., J. Med. Chem., 36:1923 (1993)). The onlyrequirement is that the oligonucleotide primer should possess a sequenceat least a portion of which is capable of binding to a portion of atarget sequence.

The target polynucleotide may be double stranded or single stranded. Insome embodiments, at least a portion of the single-stranded targetpolynucleotide is completely or substantially complementary to at leasta portion of the oligonucleotide probe immobilized on the microarray. Inother embodiments, the single-stranded target polynucleotide iscompletely complementary to the oligonucleotide probe immobilized on themicroarray.

Employing PCR, RT-PCR (for RNA molecules) or other methods,polynucleotide molecules/agents of interest can be converted to nucleicacid fragments and labeled with biotin, digoxin or the similar, whichthen binds with moieties on the surface of particles/beads. By couplingto the particles or beads, these nucleic acid fragments in solution areenriched. For double-stranded nucleic acid fragments, they are denaturedto single-stranded ones. Beads are then coupled to specific microarrayspots through target-probe hybridization, which directly or throughfurther modifications, facilitate the detection of results withnon-expensive devices or common commercial microarray scanners. Specificgenes, SNPs or gene mutations, such as deletions, insertions, andindels, are thus identified. For SNPs/mutations, they are valuable forbiomedical research and for developing pharmaceutical compounds ormedical diagnostics. SNPs are also evolutionarily stable—not changingmuch from generation to generation—making them convenient to follow inpopulation studies.

Any method may be used to assay the polynucleotide, that is, todetermine the polymorphic sites, in this step of the present disclosure.For example, any of the primer extension-based methods, ligase-basedsequence determination methods, mismatch-based sequence determinationmethods, or microarray-based sequence determination methods describedabove may be used, in accordance with the present disclosure.Alternatively, such methods as restriction fragment length polymorphism(RFLP) detection, single strand conformation polymorphism detection(SSCP), denaturing gradient gel electrophoresis (DGGE), denaturinghigh-performance liquid chromatography (DHPLC), PCR-based assays such asthe Taqman® PCR System (Applied Biosystems) may be used.

Allele-specific PCR (ASPCR) is known as amplification refractorymutation system (ARMS) or PCR-sequence specific primer (PCR-SSP), etc.With high accuracy, ASPCR is suitable for analyzing known SNPs/mutationsin genetic sequences, which uses DNA polymerase without the 3′-5′exonuclease activity so that if the 3′ end of a specific primer does notmatch the template, the primer can not be elongated and the PCR reactionis blocked. Utilizing multiplex PCR, multiple loci can be amplifiedsimultaneously, and then distinguished by DNA microarray. The PCRamplification may be conducted in one tube, or in different tubes.

By employing the universal array technology, Tag sequences areconjugated with primers, and their final products can readily hybridizewith the Tag probes. Microarrays here just serve as a decode tool. TheTag sequences are artificially designed and subject to criticalfiltering, they have the corresponding complementary sequences, cTagsequences. Each combination of Tag and cTag corresponds to an allele ofa SNP/mutation in the target gene. The Tm difference between differentTag sequences equals or is less than 5° C., and the Tag sequences haveno cross-hybridization among themselves or with the group of primers,have low homology to the species of the sample genomic DNA, and nohair-pin structures. Determination of genes or genotypes is based on thehybridization signal and the position of the Tag probes on microarrayhybridized with the PCR products.

For the universal tag array, one can use many more or less Tag sequenceswith or without replicate spots for specific applications. These Tagsequences may be designed by methods of bioinformatics. Tag probes canalso be derived from a biological species different from the species ofthe target gene. For example, if the species of the target is fromhuman, the Tag sequences can be derived from sequences of bacteria. Inone aspect, the Tag sequence is single stranded oligonucleotide orpeptide oligonucleotide.

In one aspect, the universal array disclosed herein is different fromthe common microarray. For common microarray, the probes on the arraymay be gene-specific or allele-specific oligonucleotides. Differenttarget gene panel or SNP/mutation panel needs different format ofmicroarray. However, the universal array in the present disclosurecomprises Tag probes which are specifically designed, so they are notassociated with allele-specific oligonucleotides or primers. The Tagsequences can be used as codes for different SNP/mutation of differentgenes or different species. One format of universal array can be usedfor detection of any gene or genotype. So such array is universal andthe process of detection is a de-coding step.

H. Kits

A kit useful for labeling a target molecule with a luminophore fordetecting the target molecule using a microarray is provided in thepresent disclosure. In certain aspect, the present disclosure is alsoembodied in a kit comprising a universal Tag array. Preferably, the kitof the present disclosure comprises set of primers for ASPCRamplification of a genetic information comprising two allele-specificprimers and a common primer as set forth in Table 2. The kit of thepresent disclosure may also comprise a polymerizing agent, for example,a thermostable nucleic acid polymerase such as those disclosed in U.S.Pat. Nos. 4,889,818; 6,077,664, and the like. The kit of the presentdisclosure may also comprise chain elongating nucleotides, such as dATP,dTTP, dGTP, dCTP, and dITP, including analogs of dATP, dTTP, dGTP, dCTPand dITP, so long as such analogs are substrates for a thermostablenucleic acid polymerase and can be incorporated into a growing nucleicacid chain. In a preferred embodiment, the kit of the present disclosurecomprises at least one oligonucleotide primer pair, a polymerizingagent, and chain elongating nucleotides. The kit of the presentdisclosure may optionally include buffers, vials, microtiter plates, andinstructions for use.

In some embodiments, the kit comprises a means for labeling a subset ofa plurality of target molecules with a luminophore. In some embodiments,a luminophore is attached chemically to aid in the labeling anddetection of a biomolecule such as a protein, antibody, or amino acid.In some embodiments, the luminophore binds to a specific region orfunctional group on the target molecule and can be attached chemicallyor biologically. Various labeling techniques such as enzymatic labeling,protein labeling, and genetic labeling can be utilized.

In other embodiments, the kit comprises a means for labeling a subset ofthe plurality of functional moieties with a luminophore. In someembodiments, the kit comprises a means for labeling the labelingmolecule with a luminophore. In some embodiments, the kit comprises ameans for labeling the binding molecule with a luminophore. Labeling ofthe luminophore to the target molecule, the functional moiety, theparticle, the labeling molecule, or the binding molecule can be directlabeling or indirect labeling, and can be by covalent binding ornon-covalent binding. In some embodiments, the luminophore can beattached chemically or biologically. Various labeling techniques such asenzymatic labeling, protein labeling, and genetic labeling can beutilized.

In some embodiments, the kit comprises a means for introducing theluminophore into or onto the particle. For example, the luminophore canbe mixed with the material of a microparticle before, during, and/orafter the formation of the microparticle. The interaction between theluminophore and the particle can be direct or indirect, and can be bycovalent binding or non-covalent binding. In some embodiments, theluminophore is introduced into or onto the particle by passivediffusion, active targeting (for example, via a receptor-ligandinteraction), mechanical mixing, electrophoresis, or magneticinteraction between the luminophore and the particle.

In some embodiments, buccal swabs and dried blood spots from familiesaffected by deafness are collected, and DNA is extracted and subjectedto a method disclosed herein. Thus, in certain aspects, a kit disclosedherein further comprises a means for extracting DNA from a sample, forexample, a biological sample. As used herein, a “biological sample” canrefer to any sample obtained from a living or viral (or prion) source orother source of macromolecules and biomolecules, and includes any celltype or tissue of a subject from which nucleic acid, protein and/orother macromolecule can be obtained. The biological sample can be asample obtained directly from a biological source or a sample that isprocessed. For example, isolated nucleic acids that are amplifiedconstitute a biological sample. Biological samples include, but are notlimited to, body fluids, such as blood, plasma, serum, cerebrospinalfluid, synovial fluid, urine and sweat, tissue and organ samples fromanimals and plants and processed samples derived therefrom. Methods forDNA extraction from biological samples are known in the art.

I. Exemplary Embodiments

The following examples are offered to illustrate but not to limit thepresent disclosure.

Microarray-based assay integrated with paramagnetic microspheres wasused for multiplexed analysis of SNPs/mutations related to hereditaryhearing loss. Commercial fluorescent scanner was employed to detect theresults, which were accomplished by enriching multiple PCR products withmicrospheres, harvesting ssDNA fragments, coupling microspheres touniversal Tag array through hybridization, and decoding them with theuniversal Tag array.

The Tag probes on the universal array are designed according to theformat: NH₂-TTTTTTTTTTTTTTT-TagX, where X is a natural number between 1and 12. The Tag probes have a 5′-amino group modification, followed bypoly-dT15, followed by Tag1 to Tag12 with the sequences 1 to 12 listedin Table 1, respectively. The nucleotide sequences of Tag1 to Tag12 inthe Tag probes are identical to the corresponding sequences of Tag1 toTag12 of the primers, respectively.

TABLE 1 The probes of the universal Tag array Name Sequence (5′→3′)Structure Tag-1 NH₂-T15-GAGGAGATCGTAGCTGGTGCAT NH₂-T15- (SEQ ID NO: 1)Tag1 Tag-2 NH₂-T15-TCGCTGCCAACCGAGAATTGCA NH₂-T15- (SEQ ID NO: 2) Tag2Tag-3 NH₂-T15-GAGCAAGCGCAAACGCAGTACT NH₂-T15- (SEQ ID NO: 3) Tag3 Tag-4NH₂-T15-GCATAGACGTGGCTCAACTGTC NH₂-T15- (SEQ ID NO: 4) Tag4 Tag-5NH₂-T15-CAAGGCACGTCCCAGACGCATCAA NH₂-T15- (SEQ ID NO: 5) Tag5 Tag-6NH₂-T15-TCGGCACGCGCGAGATCACCATC NH₂-T15- (SEQ ID NO: 6) Tag6 Tag-7NH₂-T15-TTTTCCCGTCCGTCATCGCTCAAG NH₂-T15- (SEQ ID NO: 7) Tag7 Tag-8NH₂-T15-GGTATCGCGACCGCATCCCAATCT NH₂-T15- (SEQ ID NO: 8) Tag8 Tag-9NH₂-T15-TCCCTGTCTCGTTGCGTGTCTCGT NH₂-T15- (SEQ ID NO: 9) Tag9 Tag-10NH₂-T15-GTTAGGGTCGCGCCAAACTCTCC NH₂-T15- (SEQ ID NO: 10) Tag10 Tag-11NH₂-T15-AGCTAGACCACTCAGCAGACTG NH₂-T15- (SEQ ID NO: 11) Tag11 Tag-12NH₂-T15-CGCCTTAGACAGCTTGCTCATG NH₂-T15- (SEQ ID NO: 12) Tag12

The probes in Table 1 were dissolved in sample buffer, printed on thesurface of aldehyde-coated glass slides, and arranged as shown in FIG.1A. The probes shown in darker color are used to detect correspondingwild-type alleles, and the other probes are used to detect correspondingmutant alleles, and each probe is repeated 3 times (i.e., in three rowson the microarray).

As shown in FIG. 1, the corresponding polymorphism sites detected by theprobes in FIG. 1A are shown in FIG. 1B. In FIG. 1B, W indicates theprobe is for the wild-type allele of the polymorphism site, and Mindicates the probe is for the mutant allele of the polymorphism site.

The polymorphism sites are as follows:

The polymorphism sites c.35delG, c.176_191del16, c.235delC andc.299_300delAT are located on GJB2 (Cx26) gene (NM_004004.5, GI:195539329).

The polymorphism site c.2168A>G is located on SLC26A4 (PDS) gene(NM_000441.1, GI: 4505696).

The polymorphism site m.1494C>T is located on 12S rRNA (MTRNR1,belonging to the mitochondrial genes) gene (NC_012920, GI: 251831106).

Wild-type or mutant polymorphism sites are as follows:

The mutation of “c.35delG” is located on the GJB2 gene. From the 5′ endof the coding region, if the 35th nucleotide G is deleted, the allele isa c.35delG mutant. Otherwise the allele is wild-type.

The mutation of “c.176_191del16” is located on GJB2 gene. From the 5′end of the coding region, if the 16 consecutive nucleotides from the176th nucleotide to 191th nucleotide are deleted, the allele is ac.176_191del16 mutant. Otherwise the allele is wild-type.

The mutation of “c.235delC” is located on GJB2 gene. From the 5′ end ofthe coding region, if the 235th nucleotide C is deleted, the allele is ac.235delC mutant. Otherwise the allele is wild-type.

The mutation of “c.299_300delAT” is located on GJB2 gene. From the 5′end of the coding region, if the 2 consecutive nucleotides from the299th nucleotide to 300th nucleotide are deleted, the allele is ac.299_300delAT mutant. Otherwise the allele is wild-type.

The mutation of “c.2168A>G” is located on SLC26A4 gene. From the 5′ endof the coding region, if the 2168th nucleotide A is mutated to G, theallele is a c.2168A>G mutant. Otherwise the allele is wild-type.

The mutation of “m.1494C>T” is located on the 12S RNA gene. If the1494th nucleotide C is mutated to T, the allele is a m.1494C>T mutant.Otherwise the allele is wild-type.

In FIG. 1B, 35W indicates the wild-type probe of c.35delG (Tag1), 176Windicates the wild-type probe of c.176_191del16 (Tag3), 235W indicatesthe wild-type probe of c.235delC (Tag5), 35M indicates the mutant probeof c.35delG (Tag2), 176M indicates the mutant probe of c.176_191del16(Tag4), 235M indicates the mutant probe of c.235delC (Tag6), 299Windicates the wild-type probe of c.299_300delAT (Tag7), 2168W indicatesthe wild-type probe of c.2168A>G (Tag9), 1494W indicates the wild-typeprobe of m.1494C>T (Tag11), 299M indicates the mutant probe ofc.299_300delAT (Tag8), 2168M indicates the mutant probe of c.2168A>G(Tag10), 1494M indicates the mutant probe of m.1494C>T (Tag12). Namewith the ‘W’ or ‘M’ suffix represents the probe corresponding to thewild-type or mutant allele at the SNP/mutation locus, respectively. Tags1-12 in FIG. 1A correspond to the above Tags 1-12, respectively.

Multiplex PCR primers used for analyzing a total of 6 SNPs/mutations arelisted in Table 2. In column Mutation Type ‘del’ represents a deletionmutation, e.g., c.35delG means a deletion of G at position 35 in thecoding region of GJB2; ‘>’ represents a substitution mutation, e.g.c.2168A>G means a substitution of A by G at position 2168 in the codingregion of SLC26A4 (PDS). Primer Name with ‘WT’ or ‘MU’ suffix representsan allele-specific primer capable of specifically amplifying thewild-type or mutant allele at the SNP/mutation locus, respectively.Primer Name with a ‘RB’ suffix represent a common primer, biotinylatedat the 5′-termini, capable of amplifying both the wild-type allele andthe mutant allele of the target genetic fragments including theSNP/mutation locus. For each SNP/mutation locus the two allele-specificprimers respectively pair with the common primer.

TABLE 2 Hereditary deafness related SNP/ mutation and primers MutationPrimer Name Primer Sequence (5′→3′) c.35delG t35delG-WTTag1-TGTTTGTTCACACCCCCGAG (SEQ ID NO: 13) t35delG-MUTag2-TGTTTGTTCACACCCGCAG (SEQ ID NO: 14) 35delG-RBBiotin-GCATGCTTGCTTACCCAGAC (SEQ ID NO: 15) c.176_191del16t176_191del16-WT Tag3-CCAGGCTGCAAGAACGTGTG (SEQ ID NO: 16)t176_191del16-MU Tag4-ACCCTGCAGCCAGCTACG (SEQ ID NO: 17) 176_191del16-RBBiotin-GAGCCTTCGATGCGGACC (SEQ ID NO: 18) c.235delC t235delC-WTTag5-AAACGGCTATGGGCCCTG (SEQ ID NO: 19) t235delC-MUTag6-ATCCGGCTATGGGCCTG (SEQ ID NO: 20) 235delC-RBBiotin-GAGCCTTCGATGCGGACC (SEQ ID NO: 21) c.299_300delATt299-300delAT-WT Tag7-TGGCCTACCGGAGACATGA (SEQ ID NO: 22)t299-300delAT-MU Tag8-CGTGGCCTACCGGAGACGA (SEQ ID NO: 23)299-300delAT-RB Biotin-GAGCCTTCGATGCGGACC (SEQ ID NO: 24) c.2168A > Gt2168A > G-WT Tag9-GACACATTCTTTATGACGGTCCA (SEQ ID NO: 25) t2168A > G-MUTag10-ACATTCTTTTTGTCGGTCCG (SEQ ID NO: 26) 2168A > G-RBBiotin-CAAGGTTTTCCAGATTGCTGAG (SEQ ID NO: 27) m.1494C > T t1494C > T-WTTag11-CTTTGAAAGTATACTTGAGGAGG (SEQ ID NO: 28) t1494C > T-MUTag12-CTTTGAAGTATACTTGAGGAGA (SEQ ID NO: 29) 1494C > T-RBBiotin-CCCTGATGAAGGCTACAAAG (SEQ ID NO: 30)

For each polymorphism locus, there are two allele-specific primers and acommon biotin-labeled primer. The allele-specific primers comprise twoparts, namely, a tag sequence at the 5′ end, and a nucleotide sequencein the 5′-3′ direction that is complementary to target gene locus. Foreach SNP/mutation locus the two allele-specific primers respectivelypair with the common primer. Thus, the allele specific primers andcommon primer can be used in multiple allele-specific PCR amplificationsto amplify the polymorphism locus DNA fragments.

In Table 2, there are 18 primers that are used for multipleallele-specific PCR amplifications.

In specific examples, the MyOne Dynal magnetic beads were coated bystreptavidin (Invitrogen Dynal AS, Oslo, Norway), with particle sizebeing 1 micron in diameter. The particle coated with streptavidin can beused to capture the biotin-labeled DNA fragments.

In specific examples, the hybridization buffer was prepared as follows.The buffer comprises a solvent and a solute, the solute being SDS, andthe solvent being H₂O, SSC, Denhardt's and formamide. The hybridizationbuffer contains 0.15% SDS (0.15 g/100 ml), SSC (9×), Denhardt's (7.5×),and formamide (37.5% v/v). The hotstar polymerase and the buffer werefrom Promega Corporation, catalogue number M500X.

EXAMPLE 1 Method Of Detecting Luminophore-Labeled Target Molecules

In this example, whole genome DNA was used as the template for PCRreaction to amplify target molecules.

Step I: The genomic DNA was extracted from whole blood from individualswho have been genotyped as normal. The genomic DNA was subject tomultiple allele-specific PCR amplifications using the 18 primers listedin Table 2, to achieve the enrichment of DNA fragments at multiplepolymorphism sites.

Step II: Single-stranded DNA was prepared using the MyOne Dynal magneticbeads in accordance with the instruction manual. 3 μl MyOne Dynalmagnetic beads were pipetted into 8 μl of the amplified product, and themixture was incubated for 10 min. The magnetic beads were then treatedwith freshly prepared NaOH solution (0.1 M) for 10 min. The NaOHsolution was then removed, and 15 μl of hybridization buffer was addedto form the hybridization mixture to be applied to a microarray.

Step III: The hybridization mixture was then applied to the microarraychip, and incubated for 1 hour at 50° C. If the magnetic beads weremanipulated by magnetic force, the hybridization incubation time can bereduced to 15 min. The microarray chip was then washed with buffer I andbuffer II once each at room temperature. Wash buffer I was 1 ×PBS with0.2% Tween-20 (v/v), and wash buffer II was 0.03×SSC. The chip was thendried by centrifugation, and scanned with a commercial fluorescentmicroarray scanner (or eye observation). The scanner was LuxScan 10Kfrom Capitalbio Corporation, Beijing. The laser power was 90%, andphotomultiplier (PMT) was 600. The images were analyzed using SpotData(Capitalbio Corporation, Beijing).

When normal human genome DNA was used as template in the PCR, all theprimers specific for the mutation sites would not amplify, and no PCRproducts were generated that specifically hybridized to themutant-specific microarray probes. Wild-type primers would amplify thewild-type target sequences, and the PCR products after multiple roundsof amplification in the PCR reactions contained 6 kinds ofoligonucleotide fragments (corresponding to 6 wild-type sites for eachof the following loci: c.35delG, c.176_191del16, c.235delC,c.299_300delAT, c.2168A>G and m.1494C>T). These oligonucleotidefragments were hybridized with the wild-type probes on the microarraychip. The oligonucleotide fragments coupled to the microparticles werelabeled with a luminophore according to the examples below, andsubjected to hybridization on the microarray to probes that correspondedto the 35W, 176W, 235W, 299W, and 1494W positions. Throughhybridization, the oligonucleotide fragments labeled with theluminophore were immobilized on the microarray chip, and the fluorescentsignals were detected by the scanner at the positions where specifictarget-probe hybrization took place.

EXAMPLE 2 Labeling Target Molecules By Labeling A Subset Of TargetMolecules With A Luminophore

In this example, a plurality of target molecules were labeled bylabeling one type of target molecule in the plurality of targetmolecules. The PCR primers used in this example were identical to thosein Table 2, except that the primer 35delG-RB was labeled with theluminophore Cy3 (identified by * in Table 3).

TABLE 3 Primer 35delG-RB labeled with luminophore Mutation Primer NamePrimer Sequence(5′→3′) c.35delG t35delG-WT Tag1-TGTTTGTTCACACCCCCGAG(SEQ ID NO: 13) t35delG-MU Tag2-TGTTTGTTCACACCCGCAG (SEQ ID NO: 14)35delG-RB Biotin-GCAT*GCTTGCTTACCCAGAC (SEQ ID NO: 15)

The PCR primers were used to amplify and enrich the polymorphism siteDNA fragments using normal human genome DNA as template. The PCRreaction system was set up according to Table 4, and the PCRamplification cycle is shown in Table 5.

TABLE 4 PCR system Volume Reagent Name (μl) Concentration ddH₂O 13.4 /2.5 mM dNTP 2 0.2 mM 5 × buffer 5 1 × buffer 25 mM MgCl₂ 1 1 mM Hotstarpolymerase (5 U/μl) 0.2 0.04 U/μl specific primers mix 1.2 1.2 μM (thesame concentration) common primers mix 1.2 1.2 μM (the sameconcentration) Human DNA (10 ng/μl) 1 0.4 ng/μl total 25 /

TABLE 5 PCR Amplification Cycle Temp. (° C.) 95 95 55 72 72 12 time (s)600 30 15 130 600 forever cycle 1 35 1

The MyOne Dynal magnetic beads used in this example were coated bystreptavidin. Preparation of single-stranded DNA and hybridization tothe microarray were performed according to Step II and Step III ofExample 1 above. The results of microarray chip scanning are shown inFIG. 2.

The arrangement of probes in FIG. 2 corresponds to the location ofprobes in FIG. 1A or FIG. 1B. The hybridization signals were detected inthe first and third lines. For example, in the arrangement of probesshown in FIG. 1B, only the probes in the first and third lines (thewild-type probes) emitted specific hybridization signals, as shown inFIG. 2.

In this example, the target molecules were labeled with the luminophore,wherein only a subset of the target molecules was directly labeled withthe luminophore. The results in FIG. 2 indicate that such a labelingmethod (as illustrated in FIG. 3) can be used to label a plurality oftarget molecules for them to be detected and analyzed using a microarraychip.

EXAMPLE 3 Labeling The Surface Of A Particle With A Luminophore

In this example, the Streptavidin moieties on the surface of theparticle were labeled with the luminophore Cy3.

The PCR primers listed in Table 2 were used to amplify and enrich thepolymorphism site DNA fragments using normal human genome DNA astemplate, by allele-specific PCR. The PCR reaction system was set upaccording to Table 4, and the PCR amplification cycle is shown in Table5.

Preparation of single-stranded DNA and hybridization to the microarraywere performed according to Step II and Step III of Example 1 above. Theresults of microarray chip scanning are shown in FIG. 4.

The arrangement of probes in FIG. 4 corresponds to the location ofprobes in FIG. 1A or FIG. 1B. The hybridization signals were detected inthe first and third lines. For example, in the arrangement of probesshown in FIG. 1B, only the probes in the first and third lines (thewild-type probes) emitted specific hybridization signals, as shown inFIG. 4.

In this example, the luminophore was used to label the Streptavidin onthe surface of the microparticles. The results in FIG. 4 indicate thatsuch a labeling method (as illustrated in FIG. 5) can be used to label aplurality of target molecules for them to be detected and analyzed usinga microarray chip.

EXAMPLE 4 Labeling A Microparticle By Introducing A Luminophore Into OrOnto The Microparticle

In this example, the luminophore Cy3 was introduced into or ontomicroparticles coated with Streptavidin. In one experiment, theluminophore Cy3 was mixed into the microparticles, wherein themicroparticles were coated with Streptavidin.

The PCR primers listed in Table 2 were used to amplify and enrich thepolymorphism site DNA fragments using normal human genome DNA astemplate, by allele-specific PCR. The PCR reaction system was set upaccording to Table 4, and the PCR amplification cycle is shown in Table5.

Preparation of single-stranded DNA and hybridization to the microarraywere performed according to Step II and Step III of Example 1 above. Theresults of microarray chip scanning are shown in FIG. 6.

The arrangement of probes in FIG. 6 corresponds to the location ofprobes in FIG. 1A or FIG. 1B. The hybridization signals were detected inthe first and third lines. For example, in the arrangement of probesshown in FIG. 1B, only the probes in the first and third lines (thewild-type probes) emitted specific hybridization signals, as shown inFIG. 6.

In this example, the luminophore was introduced into or onto themicroparticles. The results in FIG. 6 indicate that such a labelingmethod (as illustrated in FIG. 7) can be used to label a plurality oftarget molecules for them to be detected and analyzed using a microarraychip.

EXAMPLE 5 Using A Labeling Molecule To Label A Plurality Of TargetMolecules

In this example, a labeling molecule SP1 was synthesized and used tolabel a plurality of target molecules. The sequence of SP1 is shown inTable 6.

TABLE 6 Sequence of labeling molecule SP1 Name Sequence (5′→3′) SP1Biotin-*GCACGCTATCACGTTAGAC (SEQ ID NO: 31)

The luminophore Cy3 was used to label the dGTP in the sequence of SP1,identified by * in Table 6.

The PCR primers listed in Table 2 were used to amplify and enrich thepolymorphism site DNA fragments using normal human genome DNA astemplate, by allele-specific PCR. The PCR reaction system was set upaccording to Table 4, and the PCR amplification cycle is shown in Table5.

In this example, the magnetic beads were coated by Streptavidin.

Preparation of single-stranded DNA and hybridization to the microarraywere performed according to Step II and Step III of Example 1 above,except that the procedures included incubating the PCR products with thetreated microparticles (3 μL), SP1 (1 μL, final concentration 0.1 μM),and 8 μl of the amplified products for 10 minutes. The results ofmicroarray chip scanning are shown in FIG. 8.

The arrangement of probes in FIG. 8 corresponds to the location ofprobes in FIG. 1A or FIG. 1B. The hybridization signals were detected inthe first and third lines. For example, in the arrangement of probesshown in FIG. 1B, only the probes in the first and third lines (thewild-type probes) emitted specific hybridization signals, as shown inFIG. 8.

In this example, the luminophore was present on a labeling molecule SP1,which, like the biotin-labeled target molecules, specifically binds tothe streptavidin on the microparticles. The results in FIG. 8 indicatethat such a labeling method (as illustrated in FIG. 9) can be used tolabel a plurality of target molecules for them to be detected andanalyzed using a microarray chip.

EXAMPLE 6 Using A Binding Molecule To Label A Plurality Of TargetMolecules

In this example, a binding molecule C1494 was synthesized and used tolabel a plurality of target molecules. The sequence of C1494 is shown inTable 7, and the C1494 sequence is complement to sequences of productsof the m.1494C>T loci by PCR.

TABLE 7 Sequence of binding molecule C1494 Name Sequence (5′→3′) C1494ACT*TACCATGTTACGACTAGT (SEQ ID NO: 32)

The luminophore Cy3 was used to label the dTTP in the sequence of C1494,identified by * in Table 7.

In this example, the magnetic beads were coated by Streptavidin.

Preparation of single-stranded DNA and hybridization to the microarraywere performed according to Step II and Step III of Example 1 above,except that the procedures included mixing the hybridization mixturewith C1494 (final concentration 0.1 μM) before applying the mixture tothe microarray for incubation at 50° C. for 1 hr. The results ofmicroarray chip scanning are shown in FIG. 10.

The arrangement of probes in FIG. 10 corresponds to the location ofprobes in FIG. 1A or FIG. 1B. The hybridization signals were detected inthe first and third lines. For example, in the arrangement of probesshown in FIG. 1B, only the probes in the first and third lines (thewild-type probes) emitted specific hybridization signals, as shown inFIG. 10.

In this example, the luminophore was present on a binding moleculeC1494, which specifically hybridizes to a subset of target molecules ofa plurality of target molecules. The results in FIG. 10 indicate thatsuch a labeling method (as illustrated in FIG. 11) can be used to labela plurality of target molecules for them to be detected and analyzedusing a microarray chip.

The invention claimed is:
 1. A method of labeling a plurality of targetmolecules with a luminophore for detecting the target molecules using amicroarray, the method comprising: a) coupling each target molecule of aplurality of target molecules to a particle to form a target-particlecomplex, wherein each target molecule comprises a modification moietyand the particle comprises a plurality of functional moieties, whereineach target molecule is coupled to the particle via interaction betweenthe modification moiety and the functional moiety, and wherein eachtarget molecule comprises a target portion and a portion capable ofspecific binding to a probe molecule immobilized on the microarray,wherein the microarray comprises a plurality of immobilized probemolecules; and b) providing a luminophore on the target-particlecomplex, thereby directly or indirectly labeling the plurality of targetmolecules with the luminophore, wherein the providing step comprises:introducing the luminophore into or onto the particle to label theparticle, and incubating the luminophore-labeled particle with theplurality of target molecules, whereby the plurality of target moleculesare coupled to the particle, wherein the target molecule comprises afirst polynucleotide, and the probe molecule comprises a secondpolynucleotide that is complementary to the first polynucleotide.
 2. Themethod of claim 1, wherein the method further comprises a step ofdetecting the plurality of target molecules using the microarray.
 3. Themethod of claim 2, wherein the detecting step comprises measuringluminescence of the target-particle complex, wherein the complex isimmobilized on the microarray via specific binding of the targetmolecule to the immobilized probe molecule.
 4. The method of claim 1,wherein the particle diameter is between about 0.1 micrometer and about10 micrometers.
 5. A method of detecting a target molecule using amicroarray, the method comprising: a) labeling a target molecule with aluminophore according to claim 1; b) incubating the target molecule andthe particle with the microarray, wherein the target-particle complex isimmobilized on the microarray via specific binding of the targetmolecule to the immobilized probe molecule; and c) measuringluminescence of the immobilized target-particle complex, wherein theluminescence indicates the absence, presence, and/or amount of thetarget molecule.
 6. The method of claim 1, wherein a spot on themicroarray ranges from about 10 micrometers to about 5000 micrometers indiameter.
 7. The method of claim 1, wherein the target moleculecomprises a universal tag sequence, or at least two different universaltag sequences are used.
 8. The method of claim 7, wherein the T_(m)difference between different tag sequences equals or is less than about5° C.
 9. The method of claim 1, further comprising detecting the targetmolecule by device selected from the group consisting of a microarrayscanning device, a flatbed scanner, a camera, and a portable device. 10.The method of claim 1, wherein the target molecule is associated with agenetic information.
 11. The method of claim 10, wherein the geneticinformation is associated with hereditary hearing loss and/orbeta-thalassemia.
 12. The method of claim 11, wherein the geneticinformation is within a target gene of gap junction beta-2 protein(GJB2) also known as connexin 26 (Cx26); gap junction protein beta 3(GJB3); solute carrier family 26 member 4 (SLC26A4); or 12S rRNA(mitochondrially encoded 12S ribosomal RNA, MTRNR1).
 13. The method ofclaim 12, wherein: the genetic information in GJB2 is selected from thegroup consisting of c.35delG, c.176_191del1 6, c.235delC, andc.299_300delAT; the genetic information in SLC26A4 is selected from thegroup consisting of c.2168A>G, IVS7-2A>G, c.1229C>T, c.1975G>C,c.1174A>T, c.1226G>A, c.2027T>A, and IVS15+5G>A; the genetic informationin 12S rRNA is selected from the group consisting of m.1494C>T andm.1555A>G; and/or the genetic information in GJB3 is c.538 C>T.
 14. Themethod of claim 11, wherein the genetic information is within a targetgene of hemoglobin subunit beta (HBB).
 15. The method of claim 14,wherein the genetic information in HBB is selected from the groupconsisting of c.−82C>A, c.−80T>C, c.−79A>G, c.−78A>G, c.−11_8delAAAC,c.79G>A, c.91A>G, c.92+1G>T, c.92+5G>C, c.315+5G>C, c.316−197C>T,c.2T>G, c.45_46insG, c.84_85insC, c.52A>T, c.113G>A, c.126_129deICTTT,c.130G>T, and c.216_217insA.
 16. The method of claim 10, whereinallele-specific polymerase chain reaction (ASPCR) is used to amplify thegenetic information, and the set of primers for the ASPCR comprises atleast two allele-specific primers and one common primer.
 17. The methodof claim 16, wherein the allele-specific primers and the common primerhave a sequence selected from the group consisting of SEQ ID NO: 13, SEQID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18,SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO:23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ IDNO: 28, SEQ ID NO: 29, and SEQ ID NO: 30.